Nanomedicine in Cancer Therapeutics: Current Perspectives from Bench to Bedside

Abstract

Cancer is among the leading causes of death worldwide, with projections indicating that it will claim 35 million lives by the year 2050. Conventional therapies, such as chemotherapy and immune modulation, have reduced cancer mortality to some extent; however, they have limited efficacy due to their broad mode of action, often resulting in cytotoxic effects on normal cells along with the malignant tissues, ultimately limiting their overall optimal therapeutic efficacy outcomes.

Rapid advances in nanotechnology and an evolving understanding of cancer mechanisms have propelled the development of a diverse array of nanocarriers to vanquish the hurdles in achieving sophisticated drug delivery with reduced off-target toxicity. Nanoformulations can deliver the anti-cancer agents precisely to the tumor cell by integrating a multitarget approach that allows for tissue-, cell-, or organelle-specific delivery and internalization. Despite the immense interest and unmatched advancements in modern oncology equipped with nanomedicines, only a few nanoformulations have successfully translated into clinical settings. A major reason behind this shortcoming is the lack of a rationale design incorporating smart, responsive targeting features, leading to a compromised therapeutic window due to inefficient internalization or erroneous intracellular localization with unsuccessful payload release. This review aims to summarize the recent perspective of nanomedicine and its translation to clinical practice, with a particular focus on the evolution of strategies used in tumor targeting from traditional EPR-based passive mechanisms to advanced active and multi-stage approaches. We highlight the coupling of organelle-specific and stimuli-responsive nanocarriers, discuss the potential of biomimetic and cell-mediated delivery systems, and also shed light on technologies such as microfluidics, tumor-on-chip models, and AI-assisted synthesis. Finally, this review explores translational hurdles ranging from biological and manufacturing challenges to regulatory bottlenecks and outlines how innovative modeling systems and engineering solutions can bridge the gap from bench to bedside in cancer nanotherapeutics.

Introduction

Cancer remains one of the most aggressive and fatal diseases, with the second leading cause of death in the United States [1], responsible for millions of deaths around the globe [2], and the incidence is expected to rise rapidly over the next 20 years in upcoming decades, driven by certain risk factors such as, but not limited to, environmental pollution, demographic changes, and the increased prevalence of lifestyle [3]. This marks an urgent need for effective and precise therapeutic interventions to reduce the overall mortality rate related to cancer. Although there is substantial improvement in the patient survival rate because of conventional therapeutic options like surgery, chemotherapy, and radiotherapy, however they are the least effective in treating advanced metastatic cancers [4], and their clinical outcomes have been concealed due to the lower patient response rates [5, 6]. Notably, both conventional chemotherapy and immunotherapies lack specificity, often targeting tumor cells alongside normal cells, leading to undesirable and sometimes fatal side effects [7, 8]. In response, there have been significant efforts for the development of more effective and less harmful treatments, in this regard nanotechnology has emerged as a promising field, which offers tremendous solutions to the limitations of traditional therapies, that have struggled to balance efficacy and toxicity [9–11]. For instance, the life-threatening complications with immune checkpoint blockades (ICBs) limited their use for cancer therapeutics, although they showed significant potential as an anti-cancer drug; however, redesigning the delivery approach using nanoscience showed a promising and effective treatment combination of ICBs and nanotechnology [12]. Moreover, it has been reported that if nanoparticle-based immune cell death inducers (ICD) are coupled with immune checkpoint inhibitors (ICIs), then this synergy can prevent the metastatic potential and relapse of tumors. Ultimately, increasing the efficiency of the cancer immunotherapy and highlighting the potential of these nanocarriers as precise delivery agents, antigen-releasing agents, and aiding in dendritic cell activation. The use of ICD here serves the function of boosting antitumor immunity and sensitizing tumor cells to ICIs. This combination of nanoparticles (NPs) with ICD and ICIs helps to overcome the immunosuppressive nature of the tumor environment when working in synergy with one another [13].

Over the past 30 years, multifunctional nanomedicine witnessed a significant expansion of research in oncology (Table 1) (Fig. 1), using lipid-based, polymeric, and inorganic nanoparticles to target cancer cells by enhancing drug solubility and circulation, improving tumor penetration, and controlling the release of the drug, ensuring a more precise and safer delivery of treatments to tumor sites [14, 15].

Table 1.

Advancement of anti-cancer nano drugs used in various tumor models

Nano Drugs platform	Active pharmaceutical ingredients	Therapeutic mechanism	Tumor model	Ref
Liposomes-based Nanodrugs	Arg-Gly-Asp-modified PEGylated liposome-encapsulated ICG (RGD-PLS-ICG)	More accumulation of overexpressed integrin α5β1 in tumor tissues facilitates accurate resection of gastric cancers	Gastric (a heterotopic subcutaneous transplantation) tumor mouse	[16]
	Tumor-homing peptide-tagged Cationic liposome-nucleic acid (CL-NA) NPs	Targeting specific tissues and cell types via the conjugation of an affinity ligand	A mouse model of peritoneally disseminated human gastric cancer	[17]
	External beam radiotherapy (EBRT)-based Rhenium 188 (188Re)-Liposome NPs emit gamma rays for imaging and beta rays for therapy	188Re-Liposome NPs deliver high-energy radiation beams to cover both gross tumors and potential microscopic tumors	Xenografts (subcutaneous) of esophageal cancer in BALB/c nude mouse	[18]
	5-fluorouracil (5-FU) and LY294002 (LY)-loaded PEGylated nanoliposome	A receptor-mediated cellular uptake that will allow the gradual release of the drugs (5-FU and LY released in a controlled manner, with LY relatively released faster compared to that of 5-FU) in the acidic environment	Esophageal squamous cell carcinoma (ESCC) subcutaneous tumor mouse	[19]
	Thermobots with doxorubicin and attenuated Salmonella in combination with high-intensity-focused ultrasound (HIFU) heating	Thermobots that actively transport membrane-attached low-temperature sensitive liposome (LTSL) release doxorubicin and target DNA Topoisomerase II with high-intensity focused ultrasound (HIFU) heating (40–42 °C) at the tumor site	Subcutaneous tumor model with transplantation of colon carcinoma cell line in BALB/c mouse	[20]
	Folate-Liposomal 5FU	Targeted folate-liposomal 5FU exacerbates high cytotoxicity in cancer cells	Subcutaneously transplanted BALB/c mouse model (colon carcinoma)	[21]
	Curcumin analog 3,5-bis (2-fluoro benzylidene)-4-piperidone (EF24) into pegylated liposomes (Lipo-EF24)	Lipo-EF24 inhibits the activation of NF-kappa-B-alpha in Pancreatic Cancer	Subcutaneous xenografts by injecting MIAPaCa-2 cells in nude mice	[22]
	Cantharidin PEGylated liposome	Cantharidin inhibits protein phosphatase 2A (PP2A), which is essential for various cellular processes, including cell growth, differentiation, and survival	Subcutaneous HepG2 xenograft tumor model (BALB/c nude mouse) of hepatocellular carcinoma	[23]
	Glycyrrhetinic acid-modified curcumin-loaded cationic liposomes (GAMCLCL)	GAMCLCL enhances the delivery and uptake of curcumin by targeting the liver-specific receptors that bind to GA within the tumor site	Subcutaneous transplantation of H22 hepatoma cells in the right foreaxillary region of the mouse	[24]
	Oleuropein-loaded folate-modified liposomes (OL-FML)	OL-FML enhances the bioavailability of oleuropein in blood and increases the liposomal endocytosis as well as active targeting through overexpressed folate receptor binding	Subcutaneously injected 22Rv1 cells in BALB/c male nude mice	[25]
	Gold nanorods (GNRs)/Doc-liposome-RLT (low-density lipoprotein receptor (LDLR)-binding peptide) (GNRs/DocL-R)	GNRs/DocL-R exhibits dual (chemo- and thermotherapy) anticancer activity via tumor-specific release and intracellular delivery of Docetaxel at the tumor site	Subcutaneously transplanted PC-3 cells in BALB/c-nu male nude mouse	[26]
Polymeric micelles	Beta-lapachone (beta-lap) micelles	Beta-lap bio-activates by NAD(P)H: quinone oxidoreductase 1, an enzyme found specifically overexpressed in non-small cell lung cancer (NSCLC) and induces necroptosis via ROS generation, followed by hyperactivation of PARP-1	Subcutaneous A549 lung tumors in BALB/c athymic nude mice	[27]
	Mixed MPEG-PLA/Pluronic copolymer micelles	MPEG-PLA/Pluronic copolymer micelles enhance bioavailability, inhibit drug efflux transporters, and overcome multidrug resistance	Human carcinoma KB cells xenograft in BALB/c nude mouse	[28]
	Nano assemblies of Phospholipids and Doxorubicin	PEG–PE–based nanocarrier of doxorubicin increases cytotoxicity by improving the penetration of dox in NSCLC cells	Subcutaneous and orthotopic transplantation of NSCLCs in the C57BL/6 mouse model	[29]
	Self-associated docetaxel in phospholipid-based micelles	mPEG2000-DSPE micelle enhanced antitumor efficacy by increasing the aqueous solubility of DTX and the side effects of Taxotere	Subcutaneous injection of MCF-7 cells in female BALB/c nude mouse	[30]
	Camptothecin (CPT)-loaded polymeric micelles	CPT-loaded polymeric micelles prolong blood circulation and enhance the accumulation of CPT in tumors	Colon 26 cells transplanted subcutaneously into CDF1 female mouse	[31]
	Fenretinide N-(4-hydroxyphenyl) retinamide	4-HPR encapsulated in polymeric micelles sustains blood retention of 4-HPR, passive accumulation at tumor sites, and leads to superior therapeutic efficacy	Murine melanoma B16BL6 cells subcutaneously transplanted in the lower back of a C57BL/6 mouse	[32]
	Paclitaxel-loaded polymeric micelles	PTX-loaded micelles increase the systemic circulation time and slow plasma elimination rate, resulting in a greater tumor growth-inhibition effect	BALB/c mouse inoculated subcutaneously in the armpit with EMT6 breast tumor cells	[33]
	SN-38-loaded chlorin-core star block copolymer micelles	Prolonged plasma residence time of SN-38/CSBC micelles and increased tumor accumulation and dual therapy for the synergistic combination of photodynamic therapy and chemotherapy	HT-29 human colon cancer xenograft model	[34]
	Three paclitaxel (PTX) loaded P105/PCL polymeric micelles	PTX-loaded P105/PCL micelles inhibit P-gP efflux activity in taxol-resistant tumor cells	Resistant SKOV-3/PTX cells xenografted subcutaneously into the right flanks of BALB/c nude mouse (nu/nu)	[35]
	Docetaxel micelle-encapsulated by a tripodal cyclotriphosphazene amphiphile [NP(PEG750) (GlyPheLeu)2Et]3 (CP750)	Docetaxel micelle encapsulated by CP750 is extraordinarily stable enough to hold docetaxel in the micelle core with no precipitation and remarkably enhanced photo-stability	Human breast cancer (MDA-MB-231) and gastric cancer (MKN-28) cells were subcutaneously injected into the back side of the right flank of BALB/c nude mice	[36]
	pH-responsive MPEG-poly (beta-amino ester) block copolymer micelles	pH-dependent micellization–demicellization behavior, with rapid release of DOX from the micelles in weakly acidic environments (pH 6.4) but very slow release under physiological conditions (pH 7.4)	Syngeneic melanoma B16F10 cells were injected subcutaneously into the backs of male C57BL/6 mice	[37]
	Paclitaxel-loaded nanoparticles (PTX-PEG5k-CA8 NPs)	PTX-PEG5k-CA8 NPs show preferential tumor accumulation and deep penetration into tumor tissue	A subcutaneous xenograft model of ovarian cancer and an orthotopic intraperitoneal model of metastatic ovarian cancer by injecting SKOV3-luc cells	[38]
	DOX-loaded PEG(2 k)-CA(4)	PEG2k-CA4 micelles exhibit high drug-loading capacity for DOX, better stability in physiological conditions, and much slower drug release properties	Subcutaneous Raji lymphoma xenograft mouse model (athymic nude Nu/Nu strain)	[39]
Lipid–polymer hybrid Nano Drugs	Oridonin-loaded wheat germ agglutinin-modified lipid-polymer hybrid nanoparticles (WGA-LPNs)	Oridonin-loaded WGA-LPNs improve enterocyte endocytosis and oral Bioavailability of oridonin increases the anticancer efficacy	HepG2 cells subcutaneously Xenografted into the right armpit region of BALB/c nude mouse	[40]
	Colon-targeting baicalin-loaded lipid-polymer hybrid nanoparticles (LPHNPs)	Baicalin from LPHNPs is much less absorbed, less available in the blood plasma, and has maximum availability in the colon	HCT-116 cells and drug-absorption study in male albino rats	[41]
	Cabazitaxel (CTX)-loaded Polymer-lipid hybrid nanoparticles (PMONPs)	CTX-PMONPs efficiently orally deliver the anticancer drug, CTX, into the systemic circulation	A subcutaneous H22 xenograft model of murine hepatocarcinoma (KM mice with subcutaneous injection in the right oxter)	[42]
	Docetaxel (DTX) encapsulated lipid polymer hybrid nanoparticles (LPHNPs-DTX)	LPHNPs-DTX enhances accumulation at the tumor site and facilitates quick internalization of LPHNPs through endocytosis and direct inhibitory action of DTX on cancer cells by retarding microtubule formation	MDA-MB-231 cells with 50% Matrigel were injected subcutaneously into the mammary fat pad of the female BALB/c mouse	[43]
	Folate-targeted nanoparticles encapsulating Paclitaxel (Ptxl) and radioisotope yttrium-90 (90Y)	Concurrent delivery of folate-targeted Ptxl and 90Y leads to higher chemo and radiotherapeutic efficacy	A murine model of ovarian cancer peritoneal metastasis: female Nu/Nu mice were injected IP with SKOV-3 cells	[44]
	Core–shell lipid-polymer hybrid nanoparticles (CSLPHNPs) with poly (Lactic-co-glycolic acid) (PLGA) core and lipid layer containing docetaxel and inhibitor of sphingosine kinase 1 (SK1), FTY720 (fingolimod)	Steady & sustained release of both drugs combined with an excellent targeting capability and re-sensitization of the prostate cancer cells to docetaxel	Subcutaneous human Prostate cancer PC-3 xenografts were established in NOD scid gamma (NSG) immunodeficient male mice by subcutaneous injection	[45]
	An aptamer-functionalized, curcumin (CUR) and Cabazitaxel (CTX) co-delivered LPNs (APT-CUR/CTX-LPNs)	A sustained release of both CUR and CTX exhibits higher tumor accumulation in prostate cancer cells	Prostate cancer xenograft mouse model (male BALB/c nude) generated by injecting LNCaP cells into the abdominal cavity	[46]
	Paclitaxel- and triptolide-coloaded lipid–polymer hybrid nanoparticles	The co-delivery system of PTX and TL achieved by synergistic therapeutic (encapsulation and cytotoxic) efficacy	A549/PTX cells were subcutaneously injected into The right flank of BALB/c-nude mice to produce the lung tumor xenografts	[47]
	Tumor-targeting peptide iRGD with a shell-core structure for co-delivery of doxorubicin (DOX) with sorafenib (SOR) (DOX + SOR/iRGD NPs)	An extended circulation and bioavailability of DOX + SOR/iRGD NPs significantly enhanced antitumor efficiency in Hepatocellular carcinoma	HCC xenograft mouse models; xenograft tumor model by HepG2 cells implantation subcutaneously	[48]
	Oxaliplatin immunohybrid nanoparticles (OIHNPs) containing oxaliplatin and anti-TRAIL	OIHNPs effectively deliver the drugs to the tumor sites and successful uptake and internalization of oxaliplatin in colorectal cancer HT-29 cells	Xenograft HT-29 cells were subcutaneously injected into the right flank of a BABL/c male nude nu/nu mouse	[49]
Dendrimers	Anionic half-generation 3.5–6.5 poly(amidoamine) PAMAM dendrimers	selective accumulation of PAMAM dendrimers in the tumor site and decreased systemic toxicity in comparison with free cisplatin	Human ovarian tumor cell line A2780 was injected subcutaneously into the right flank of female athymic nude mice	[50]
	Doxorubicin (DOX) is loaded into L-Cysteine-modified G4.5 dendrimer (GC/DOX)	γ-radiation improves DOX release from the GC/DOX under acidic pH conditions at tumor sites	In vivo zebrafish model: Human HeLa cells injected into the yolk space of 2-day-old zebrafish embryos	[51]
	TAEA-cored peptide dendrimers (TAEA-K3K6R12-DMA)	pH-triggered enhancement in DOX internalization into cancer cells and penetration into multicellular tumor cultures upon co-incubation with TAEA-K3K6R12-DMA	The PANC-1 tumor-bearing BALB/c nude mice model was established by subcutaneous inoculation at the right flank	[52]
	Doxorubicin and borneol (BO)-based on the PAMAM G5 dendrimer (FA-BO-PAMAM/DOX)	Glioma targeted delivery of doxorubicin modified with folic acid (FA) to target tumor cells, also borneol (BO), a well-known safe material derived from traditional Chinese medicine medicine, to facilitate the BBB permeability	C6 glioma cells are implanted stereotaxically at a depth of 5 mm from the brain surface into the right forebrain of a Wistar rat	[53]
	Docetaxel (DTX) Polysorbate 80 (P80) anchored poly (propyleneimine) (PPI) dendritic nanoconjugate (DTXeP80-PPI dendrimers)	DTXeP80-PPI dendrimers possess higher targeting efficiency and biodistribution of ligand-conjugated dendrimers into the brain	U87MG cells were implanted stereotactically into the right basal ganglia field of male albino Sprague Dawley rat	[54]

Fig. 1.

Historical timeline of major events in the field of cancer nanotechnology research or cancer nanomedicine. EPR: enhanced permeability and retention, DQAsomes: dequalinium-based liposome-like aggregates, FDA: US Food and Drug Administration, Doxil: liposomal doxorubicin, Abraxane: nanoparticle formulation of chemotherapeutic drug paclitaxel

Also, regarding oral therapeutics, NPs serve to be the most versatile source of delivery as their small size can easily bypass the mucus layer and have the ability to navigate the complex gastrointestinal (GI) environment, making it easier to get endocytosed and gain entry into cells. Furthermore, they are efficient enough for localized and larger payload delivery as well [55]. Recently documented studies reported that surface modifications of NPs help in increasing their bioavailability due to enhanced mucus penetration and cellular uptake. In this context, when chitosan was used to modify the core–shell of NPs, the result was balanced mucus permeation, cellular uptake, and improved oral delivery of the drugs [56]. Despite these advancements and promising outcomes, research in nanosciences still faces many hurdles before clinical adaptation. A considerable debate exists between the several research groups due to the significant disparities between the growing number of preclinical studies developing sophisticated nanomedicines, but very few among them have been approved for clinical settings [14, 57]. This imbalance goes through many challenges [58], on the pharmaceutical side, such as nanoformulation, production, mechanisms for drug release, biodegradation, and elimination, while metastatic property and tumor perfusion, permeability, and penetration, as well as the delivery of nanomedicines to target cells, overcoming endo/lysosomal barriers, and ensuring proper intracellular processing and trafficking constitutes biological challenges. Additionally, intellectual property hurdles, such as production costs and ensuring batch-to-batch consistency, also makes a substantial contribution. Notably, for a successful clinical translation, selecting the right drug and treatment regimen for the appropriate pathological condition and patient population is the main challenge [58].

The targeting of tumor tissue by nanoparticles can be done by passive and active mechanisms [59–61]. Passive targeting exploits the unique characteristics of solid tumors, like high vascular permeability and a lack of lymphatic drainage, leading to enhanced permeability and retention (EPR) effect [62, 63], allowing passive nanoparticle accumulation at the tumor site [64, 65]. Notably, the EPR effect underpins FDA-approved nanomedicine from Doxil to Apealea, with at least 15 nanoformulations in clinical use relying on EPR-mediated passive targeting [66]. However, its heterogeneous nature results in variable outcomes [67], prompting researchers to focus on active strategies for nanomedicine-mediated tumor targeting as alternative approaches to passive mechanisms, particularly in tumors with poor EPR effect, by leveraging molecular recognition, receptor-mediated transport, or engineered responsiveness for enhanced selectivity and intracellular uptake [68, 69].

With further advancements, the active strategies for solid tumors rely on cell surface marker recognition and their binding to nanoparticles linked to ligands. These ligands can be diverse depending upon targets, including peptides, antibodies, antibody fragments, and aptamers of nucleic acids [70–72]; these moieties can bind specifically to tumor antigens present on the plasma membrane, resulting in the internalization of conjugated nanoparticles [73]. Additionally, the past 10 years have observed tremendous interest in a promising biomimetic strategy, which involves the coating of plasma membranes from various cell types like stem cells, blood cells, or cancer cells on nanoparticles, which allow them to get enriched with homotypic or heterotypic adhesive properties of source cells and resulting in efficient and specific targeting of cancer cells [74, 75]. Following the internalization of nanocarriers into the target cells, it is necessary to release the payload precisely to their subcellular sites of action, like mitochondria, lysosomes, and nuclei [76–78]. Therefore, nanoparticles designed to target specific organelles have gained enormous attention recently and are regarded as the third generation of nanotherapeutics for oncology [79, 80].

Researchers have also explored the development of nanomedicines with specific physicochemical characteristics like charge, size, and surface modifications to effectively address all three tiers of tumor targeting: tissue-level accumulation, cellular uptake, and subcellular localization. However, achieving uniform performance across these levels with a single nanoparticle design remains a significant challenge. For instance, nanoparticles having large sizes, neutral charges with protected cell- or organelle-targeting ligands are more likely to enhance blood circulation and accumulate in tumors, while nanoparticles having smaller sizes (< 20 nm), positive charges with exposed targeting ligands, are better suited for deeper tumor penetration, efficient cellular uptake, and precise subcellular localization [81, 82]. These inherent design trade-offs highlight the complexity and limitations of single-phase targeting strategies. To overcome these limitations, multi-stage tumor targeting has now emerged as a key nanotherapeutic strategy by designing and adding stimuli-responsive properties like charge conversion, ligand exposure, and size shrinkage in single nanoparticles [81, 83]. Moreover, recent literature has emphasized both the translational challenges and prospects of nanomedicine in oncology [72, 84]. In this review, we present a unique and more detailed articulation of the multi-level centric strategies involved in cancer nanomedicine. It explores tumor targeting at the cellular, subcellular, and tissue levels, tracing progress from passive to organelle-specific delivery. Additionally, this review highlights underexplored yet innovative approaches such as internalizing RGD (iRGD)-mediated penetration, biomimetic delivery utilizing immune and stem cells, and integration of cutting-edge technologies such as artificial intelligence (AI)-driven nanoparticle synthesis and tumor-on-chip platforms to enhance clinical translation.

EPR-Effect and Passive Approaches in Cancer Therapy

Recent decades have witnessed significant advancements in targeting solid tumors by improving the delivery mechanisms of nanotherapeutics. The concept of EPR first described in 1986 by Matsumura and Maeda, revolutionized tumor-targeted drug delivery. The EPR effect refers to the ability of the macromolecules to accumulate preferentially in solid tumors due to their leaky vasculature and poor lymphatic drainage. This observation brings the revolution in the tumor-targeted delivery systems for macromolecular drugs and nanomedicines [85].

The principle of the EPR effect is based on the rapid and uncontrolled growth of cancer cells, which enables them to reprogram their vasculature to replenish the increasing demands of oxygen and nutrients, through the formation of structurally abnormal tumor capillaries and blood vessels having defects in their basement membrane with discontinuous epithelium [86, 87]. The resulting aperture of endothelial cells makes leaky vasculature of tumor cells, which ultimately leads to the extravasation of nanocarriers and biological macromolecules in them, representing the “enhanced permeability” portion of the EPR effect [88]. The “retention” portion of the EPR effect is observed due to disruption in the transportation of interstitial fluid because of a deficient lymphatic drainage system in solid tumors [88], which enables the detention of macromolecules or nanocarriers in the tumor interstitium for a protracted period (Fig. 2A & B) [89, 90].

Fig. 2.

Schematic illustrations of two major types of delivery mechanisms for nanoparticles in cancer cells or tumors. The upper panels describe the passive targeting mechanism for cancer cells. A Representative architecture and blood flow mechanism in healthy tissues, where the permeability of blood vessels is very low or less, as the endothelial cells form a proper barrier, making it difficult to target. B Represents the fenestrated blood vasculature in tumor tissues, where the morphology or the architecture of endothelial cells is not intact, enhancing its permeability towards nanoparticles and easy to target. The Lower panels describe the active targeting mechanism of nanoparticles to cancer cells. C Represents the endocytosis-mediated uptake of a specific liposome nanoparticle encapsulating drug molecules. For targeting purposes, the liposome or other types of nanoparticles were anchored with receptor targeting moiety that helps to bind the nanoparticle or recognize the specific receptor present on cancer cells (for example, G-protein coupled receptors), and following binding the nanoparticle is internalized through receptor-mediated internalization and encapsulated drug(s) is delivered and released to specific cancer cells through endocytosis. D Another example of active targeting of cancer stem cells. The nanoparticles of interest can be designed with specific targeting moieties for specific cell types. For example, the cancer stem cells (CSCs) can be specifically targeted using a unique antibody or other targeting moiety against CSC surface marker(s), and the chemotherapeutic drug(s) can be conjugated to these nanoparticles and help deliver to CSCs, which are in general difficult to target with such therapies. E Different types of modifications in terms of targeting moieties could be utilized to target specific cancer cell types with a specific purpose and drug

Several factors influence the strength and effectiveness of the EPR effect, including the complex crosstalk between the stromal compartment and tumor cells, the dysregulation in the production of proangiogenic and cytokine factors, the extent of perivascular tumor growth with tumor pressure, and the density of stromal response. In addition, the physicochemical properties of nanoparticles, such as (nanometric size, time in circulation, surface chemistry, and charge), are crucial in deciding the strength and efficiency of the EPR-mediated targeting [89, 91, 92]. For instance, to get rapid renal clearance from circulation, nanoparticles should be smaller than 40 kDa. At the same time, for maximum tumor extravasation, the nanoparticles must have a size of less than 200 nm. Therefore, to achieve EPR-dependent tumor targeting, the optimal size of nanocarriers should be between 50-150 nm [93, 94]. It has also been reported by recent studies that nanoparticles having a higher aspect ratio have longer circulation times with maximum tumor accumulation because they can more effectively avoid phagocytosis [95, 96].

The surface chemistry and charge distribution are also important factors that influence the circulation time of nanoparticles. Hydrophilic, neutral surfaces—often achieved by grafting polyethylene glycol (PEG)—reduce opsonization, preventing unwanted interactions and shielding from immune detection and aggregation [97, 98]. Unfortunately, it has been reported that the first injection of PEGylated liposomes induces the accelerated blood clearance phenomenon and PEG corona. The first one is due to the formation of IgM-like PEGs-specific antibodies, which resulted in the rapid elimination along with accelerated hepatic uptake of a second dose of PEGylated liposomes. Additionally, PEG corona is also a reason for steric hindrance, which prevents efficient uptake of nanocarriers by the tumor cells [97, 99]. Thus, it is necessary to keep an optimal relationship between prolonged circulation time and better internalization while designing the nanosystems for EPR-mediated tumor targeting, and this could be achieved by keeping shorter chains of PEG along with specific ligands [99]. PEGylation of nanosystems is a widely recognized and clinically accepted tool that helps in controlling their surface characteristics and furtiveness. Moreover, it also gives the freedom to attach the chemical ligand on its surface to maximize the chances of cellular internalization [100] by utilizing the EPR-independent method of tumor targeting, which we call “active strategies,” will be discussed in the following section.

However, it has recently been recognized that there are certain limitations in the use of EPR-mediated drug delivery due to the considerable variation not only across patients but also between tumor types because of genetic and tumor microenvironment differences [67]. For instance, patients having hepatocellular or renal cell carcinoma can exhibit a highly efficient EPR effect because of the maximum vascular densities as compared to individuals having prostate or pancreatic cancer [64, 101, 102]. Moreover, a study conducted by Matsumoto et al. established that the EPR is not a static phenomenon; it changes with time and has transient features [103].

Another important limitation is the scarcity of data obtained from pre-clinical models of tumors, which can accurately replicate the solid tumor physiology in humans for the proper understanding of the EPR effect. Likewise, the highly proliferative subcutaneous xenograft model of tumors has a very high EPR effect and could provide a false imprint for nanocarrier-mediated therapeutic options that rely on EPR-mediated passive targeting, moreover, there is a limited availability of patient-oriented research data for this unique phenomenon along with its effect on drug aggregation inside the tumor [104]. Hence, the convincingness and beneficial properties of the EPR effect were successfully exploited in some patients, while not in others [105]. Therefore, it is necessary to carry out further investigations to explore the EPR effect in various human tumors for the better development and design of nanoparticles to improve therapeutic response [70, 106]. Recently, a study on the efficacy of the EPR effect by microPET/CT imaging was done by Hansen et al. through copper-64-loaded PEGylated liposomes mediated targeting of spontaneous solid tumors in dogs, revealed that few solid tumors, especially carcinomas, predominantly exhibit EPR effect, which results in higher accumulation of liposomes, however, it could not hold true for all tumors [107]. Another advanced study on patients having HER2^+ve metastatic breast cancer revealed that intratumoral transport, distribution, and pharmacokinetics of poly (d,l-lactic-co-glycolic acid)-b-poly(ethylene glycol) (PLGA-PEG) based nanoparticle could be predicted by using 30-nm carboxymethyl dextran-coated magnetic nanoparticle which is an FDA approved drug, as a surrogate companion [108]. Furthermore, a study conducted by Lee and colleagues by using HER2-targeted liposomes labeled with ⁶⁴Cu in conjugation with PET/CT imaging to examine drug accumulation in 19 patients having HER2^+ve metastatic breast cancer, showed that peak accumulation of liposomes occurred in between 1–2 days and more favorable outcomes of treatment were associated with the patients having a higher concentration of ⁶⁴Cu-liposomal deposition [109]. All these lines of research advocate that clinicians can utilize imaging techniques to preselect cancer patients showing higher EPR effects and respond well to the passive therapeutic options, thus tremendously improving the outcomes of cancer therapy. In addition to these, the augmentation of the EPR effect can also be done by employing some classical methods like a pharmaceutical-based approach, which includes the administration of angiotensin-II, bradykinin, and TNF-α, or a physical approach such as hyperthermia, radiotherapy has the potential to be exploited for the partial improvement in the EPR-mediated tumor targeting (Table 2) [67, 68, 110], but they are not enough to treat the cancer patients suffering from non-leaky tumors, and therefore they could not be benefitted from the EPR principle. Hence, the heterogeneity and complex nature of the EPR effect give rise to the EPR-independent active strategies for tumor targeting through delivery systems that are sophisticatedly engineered [68].

Table 2.

Augmentation of the enhanced permeability and retention (EPR) effect of nano drugs via vasculature or inflammatory mediators and physical approaches

Method	Example	Target	Outcome	Ref
Vascular or inflammatory mediators	Angiotensin-II	Blood vessels	Induced transient hypertension enhances the EPR-mediated accumulation of nano drugs in tumor tissues and significantly improves therapeutic efficacy in animal models and patients with advanced solid tumors	[111–113]
	Bradykinin	Arteries	Triggers arteriolar dilation, enlarges the endothelial gaps, and enhances vascular permeability by stimulating the release of nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF)	[114–116]
	Tumor necrosis factor-alpha (TNF-α)	Inflammatory mediator	Enhances vascular permeability not only through direct contraction of F-actin cytoskeletal elements to promote morphological remodeling of endothelial cells but also by elevating ROS production and modulation of expression and localization of cell–cell adhesion molecules	[117–119]
Physical approaches	Hyperthermia	Vasculature permeability	To increase the pore cutoff size in tumor vessels to > 400 nm, allowing all tested liposomes (ranging from 100 to 400 nm) to extravasate into the tumor interstitium	[120–122]
	Radiotherapy	Interstitial pressure and vascular permeability	Improves intratumoral nanoparticle distribution by modulating the morphology and function of the tumor vascular and interstitial	[123–125]
	Sonoporation	Cell membranes or blood vessel walls	Ultrasound-guided microbubble destruction enhances the extravasation and accumulation of nanomedicines in tumors	[126–128]

Active Strategies for Tumor Tissue Targeting: Beyond the EPR Effect

Active strategies for nanomedicine-mediated tumor targeting have become an alternative method to passive mechanisms [129]. They are essential in increasing the therapeutic efficacy with limited off-targeting, as they precisely release the payload of genes, drugs, and theranostics to the region of interest without damaging surrounding healthy tissues and adjacent tissues. Moreover, this EPR-independent tumor targeting mechanism substantially increases the quantity and efficacy of drugs that are delivered to the target compartment in the tumor microenvironment by decorating the surface of nanoparticles with ligands like antibodies, peptides, aptamers and nucleic acids specific to the highly expressed receptors of cancer cells (Fig. 2C- E) [91, 130–132]. For instance, certain tumor cells overexpress surface markers like vascular endothelial growth factor receptor-2 (VEGFR2), CD105, and integrins αvβ3. Targeting these markers with drug-loaded nanocarriers can improve the precision of tumor cell internalization [133]. However, a mechanistic model developed by Schmidt et al. suggests that nanosystems with diameters greater than 50 nm do not show a significant increase in tumor internalization compared to non-targeted systems [134].

Exploring the Tumor Vasculature

Angiogenic characteristic of the cancer cell is driven primarily by the complex signaling cascade of the VEGF/VEGFR axis that promotes tumor growth and metastasis by forming new blood vessels. VEGF-A and its circulating isoforms VEGF121 and VEGF165 regulate these processes through the VEGFR2 receptor present on endothelial cells and are considered to be essential for sprouting angiogenesis [135]. In a glioblastoma-carrying mouse model, targeting VEGFR2 has demonstrated promising results. Here, the nano-conjugate VEGF₁₂₁-mesoporous silica was able to selectively deliver sunitinib tyrosine kinase inhibitor to tumor vasculature. The efficiency of this nano-formulation was found to be superior when compared to non-targeted formulas [136]. On the other hand, when cancer models were tested with the VEGFR2-directed mesoporous silica nanoparticles [137] and VEGF₁₂₁-functionalized nanographene oxide, both the constructs displayed enhanced tumor imaging potential, better radiotherapy efficacy, and better therapeutic delivery efficiency in mice having a human anaplastic thyroid tumor [138]. In addition, the integrin αvβ3 receptor, which is found overexpressed on active endothelial cells and several cancerous tumors [139], is emerging as a promising receptor for scientific interest. This receptor is specific for ligands with arginine-glycine-aspartic acid (RGD) motif, therefore, acting as a targeted nanoparticle tag [140, 141]. For instance, cyclic peptide RGDfK, when attached to nanoparticles, was able to effectively deliver DOX to αvβ3-expressing vasculature, resulting in significantly enhanced anti-metastatic potential in renal carcinoma [142]. In a similar study, αvβ3-targeted ⁶⁴Cu-radiolabeled hollow mesoporous silica nanoparticles have been used for PET imaging along with the guided release of sunitinib at the tumor site [143]. Likewise, another report established that RGD-grafted PLGA nanoparticles have successfully delivered paclitaxel with enhanced anti-cancer efficacy in tumor-bearing mice models [144]. Furthermore, endoglin-CD105, which is a part of the TGF-β receptor complex, is a reliable marker for tumor angiogenesis because it is expressed on proliferating endothelial cells [145–147]. By using anti-CD-105 monoclonal antibodies, nanoparticles targeted towards CD-105 were developed for imaging tumors and drug delivery. This approach has achieved promising results in several tumor models in mice [148–151]. CD-105, when modeled as an aptamer conjugated to fluorescent silica nanoparticles, was able to precisely target tumor vasculature along with enhanced therapeutic and imaging abilities [152]. The above-mentioned approaches have significant strengths in advancing cancer imaging and therapeutics by selective targeting of tumor-associated angiogenic receptors. However, it is indeed difficult to target the tumor vasculature with the help of nanomedicines owing to the abnormal function and structure of tumor blood vessels. This abnormality of tumor blood vessels results in inconsistent perfusion and doesn’t allow uniform distribution of nanoparticles. Apart from this, the restricted penetration of NPs is also a result of heterogenous tumor microenvironment, phenomena like the vasculogenic mimicry (formation of vessel-like structures by tumor cells which is independent of endothelial cells), hypoxic regions which limits the efficacy of therapies targeting vasculature thereby impairing drug activation. In a recent study it was concluded that even with the use of VEGFR2-targeted nanocarriers, the heterogenous intratumoral microenvironment is another reason for differential accumulation of NPs and therapy response [153]. Thus, to target the complex tumor vasculature, it is important to combine strategies that are able to modulate the immune system along with responding successfully to the hypoxic tumor microenvironment.

Internalizing RGD Peptide-Mediated Active Killing

Another active strategy of targeting vascular tumor endothelial cells through nanoparticles is based on the internalizing RGD peptide (iRGD), which is far better than RGD-containing peptide in penetrating tumor tissue [154]. iRGD has used the process of transcytosis to penetrate and accumulate inside the tumor, independent of drug conjugations [155], advocating its potential to be used as a therapeutic option in modern oncology. Highly expressed integrin receptors ανβ3 and ανβ5 have been used by the CendR motif of iRGD to target the cell surface and tumor vascular endothelial cells [156]. This interaction of the iRGD peptide with the target receptors leads to the proteolytic cleavage, resulting in the formation of the CRGDK/R fragment having the binding affinity for neuropilin-1 & 2 (NRP-1 & 2) to initiate the process of drug transport inside the tumor [156, 157]. Interestingly, this phenomenon of transcytosis is believed to be different from the clathrin, dynamin-mediated endocytosis mechanism because nutrient supply and mTOR signaling can affect this pathway. For example, nutrient deprivation conditions, like amino acid or glucose deficit, lead to the enhanced internalization of the CendR motif of iRGD [157]. Owing to the specific characteristic of tumor penetration, the iRGD peptide has been frequently used to improve the therapeutic index of chemotherapy drugs by reducing the toxic side effects. For instance, a study conducted by Hou et al. on a murine orthotopic breast cancer model established that phosphate-platinum complexation of iRGD on micelle-coated cisplatin enhances tumor accumulation with an excellent inhibition of tumor growth and also improved safety profile [158]. In another study, Liu et al. and others reported that the internalization of silicasome by pancreatic cancer cells has been enhanced when co-administered with iRDG in patient-derived xenografts (PDXs) [159, 160]. Another advantage of using iRGD-mediated drug delivery is its extraordinary tumor-penetrating property in cancers having very little EPRT effect, which results in effective drug targeting. For instance, pancreatic ductal adenocarcinoma could not be targeted by exploiting the EPR effect due to the dense pressure tissue along with strong mesenchymal adhesion and proliferation. For the first time, it has been observed in the orthotopic pancreatic cancer model that an irinotecan-loaded silicon nanoparticle of 130 nm in size was efficiently transported to the tumor site [159]. Similarly, in another study conducted by Akashi et al. on the KPC-PDAC model, cotreatment of iRGD along with gemcitabine remarkably inhibits the metastasis with a significant increase in the survival time [161]. It has also been reported that iRGD decoration can be used to increase the efficacy and nontoxic delivery of small interfering RNA by overcoming the matrix barrier of PDAC, resulting in the excellent inhibition of pancreatic cancer through Kras gene silencing [162]. Simultaneously, the use of iRGD also improves the tumor-penetrating capacity of nanoparticles in HepG2 cell lines and effectively silences the disease-related gene Plk1, thereby boosting the efficacy of cancer treatment [163]. More recently, a study conducted on glioblastoma by using a solid lipid nanosystem coated with iRGD having EGFR and Si-RNAs against PD-L1, showed significant improvement in the brain tumors, stimulating immunomodulation, leading to an increase in mouse survival with inhibited tumor proliferation [164]. The idea of achieving effective tumor killing by iRGD peptides-mediated NPs face several challenges. The major challenge is the heterogenous expression of neuropilin1/NRP-1 across the tumor tissues. This expression results in impaired CendR pathway which is required for effective and deep tumor tissue penetration. In addition, the exposure of CendR motif by proteolytic activation step can be inefficient in certain tumor microenvironments, giving rise to impaired internalization. For instance, study conducted by Lo et al. 2018 demonstrated that while iRGD-guided nanocomplexes improved siRNA delivery in pancreatic tumors, however, therapeutic outcomes has compromised significantly due to variability in receptor expression and tumor architecture [162]. These findings underscore the need for companion diagnostics and adaptive delivery strategies to overcome biological barriers limiting the uniform and effective action of iRGD-based nanotherapeutics.

Cellular Hitching

Cellular hitchhiking is also an important active strategy that can be employed for drug delivery systems by enveloping nanocarriers within the immune-invasive and tumor-oriented living cells like stem cells, leukocytes, and red blood cells (RBCs) (Fig. 3) [165]. Leukocytes have been considered “Trojan Horses” for the nanoparticle delivery system [166], due to their migration ability to the sites of acute inflammation by following chemoattractant gradients; they can also penetrate deep in the hypoxic tumor regions, and they can engulf pathogens through phagocytosis [167]. Monocytes and Macrophages, the most plastic type of cells in the hematopoietic system [168], have been extensively used for the internalization of doxorubicin (DOX)-loaded PLGA and reduced graphene oxide nanoparticles [169, 170], as well as nanosystems having the payload of INCB024360, an inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1) [171], to effectively target solid tumors (Fig. 4). Furthermore, enhanced accumulation at the tumor site has been observed in DOX-containing liposomes or chitosan-based micelles after encapsulation within monocytes/macrophages [172, 173]. Beyond monocytes and macrophages, other leukocytes, like the most abundant circulating and tumor-infiltrating neutrophils [174], which can cross the blood–brain barrier [175], and T cells, essential in adaptive immunity, are considered to be instrumental in tumor elimination [176], have been leveraged for nanoparticle delivery to tumor tissues. According to recent reports, post-surgical glioma has been effectively suppressed in mice by using neutrophils to deliver paclitaxel (PTX)-loaded liposomes to the brain [177]. Moreover, highly efficient and effective tumor targeting has been achieved in neutrophil-mediated transport of short-circulating magnetic nanoparticles [178]. It is globally accepted that immunotherapies are increasingly potent clinical options for cancer treatment [179], though T-cell-based immunotherapies have one limitation of reduction in T-cell activity due to the persistent antigen exposure in the TME, which leads to T-cell exhaustion, characterized by the upregulation of inhibitory receptors such as PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT, resulting in impairment of T-cell proliferation, cytokine production, and cytotoxic functions, ultimately reducing therapeutic efficacy. Furthermore, mitochondrial biogenesis of T-cells can also be repressed by TME, leading to metabolic insufficiency and dysfunction, eventually compromising T-cell responses [180]. These factors collectively contribute to the limited success of T-cell-based therapies in solid tumors [181, 182]. Researchers resolved this problem and enhanced the outcome of cancer immunotherapy by boosting the anti-cancer response of T-cells through T-cell-mediated delivery of adjuvant-loaded nanoparticles. Like, a significant increase in T-cell expansion has been observed in tumor-bearing mice when T cells conjugated with nanogels having IL-15 super-agonist complexes (IL-15Sa) have been used for solid tumor targeting as compared to systemic injection of IL-15Sa [183]. This approach of improving the efficacy and safety profile of human chimeric antigen receptor (CAR)-T cell therapy is now under clinical evaluation for various solid tumors and lymphomas [184]. Apart from leukocytes, mesenchymal stem cells (MSCs), due to their innate nature of tumor tropism, showed promising results in cell-based targeted delivery of nanoparticles [185, 186]. Several chemokines released by the tumor cells, like SDF-1, platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), interact with their specific receptors expressed on MSCs and govern their homing profile [187]. The most important reason for the recurrence of cancer is the presence of stem cells around the invasion zone of the tumor, which is difficult to target by traditional chemotherapy options. Therefore, various studies have been performed by leveraging the homing ability of MSCs to produce stem cell membrane-coated specific nano drug carriers with improved therapeutic index [188]. For instance, Zhang et al. had shown that malignant bone tumors can be treated by using polydopamine (PDA) NPs (PDA@SCM NPs) coated with membrane derived from MSC, having significant compatibility with blood circulation and slow release of drug SN38 [189]. Similarly, a study conducted by Wang et al. established that only 1/8TH dose of docetaxel-loaded PLGA nanoparticles is enough when encapsulated in MSCs to accumulate in the tumor site of lung cancer [185]. Being the primary constituent of blood [190], and due to the expression of self-markers like CD47, which helps in reducing the uptake of nanoparticles by the reticuloendothelial system, red blood cells (RBCs) are considered to be an excellent platform for nanoparticle coating for tumor-targeted delivery [191, 192]. The expression of transmembrane protein CD47 has an extracellular domain that resembles the immunoglobulin variable region (IgV) [193]. This resemblance helps in the survival of RBC-coated nanoparticles by inhibiting the macrophage-mediated phagocytosis due to the interaction of CD47 with signal-regulatory protein alpha on phagocytic cells, which recognizes CD47 as a "don’t eat me" signal, resulting in a significant increase in the half-life time with prolonged blood circulation [194–196]. For instance, mesoporous silica nanoparticles of doxorubicin encapsulated in an RBC membrane having an iRGD ligand have been developed by Huang et al. to evade and escape the immune cell-mediated phagocytosis to achieve effective tumor targeting [197]. Similarly, Sun et al. demonstrated that specific tumor targeting has also been achieved by camouflaging doxorubicin-loaded ICG-bovine serum albumin nanocomplexes in RGD-modified RBCs [198, 199]. A recent study on a pulmonary metastatic model of aggressive melanoma has opened new vistas in the usage of RBC-based nanocarriers for targeted cancer therapy, by highlighting that RBC hitchhiking could significantly increase the accumulation of nanoparticles in the lung and dramatically reduce tumor nodes [200].

Fig. 3.

Representative method to utilize specific cell types as a carrier of anticancer nanomedicines to cancer cells. Common chemical modifications in antisense oligonucleotides and RNA oligonucleotides. A Mesenchymal stem cells (MSCs) have propensity to specifically accumulate in cancer cells or show tumor tropism. B MSCs were routinely cultured and could be utilized as a nanomedicine carrier. Under specific culture conditions, these cells act as a promising host to be loaded with nanodrug/nanoparticles of interest and allowed to grow. C-G The modified MSCs can be delivered to specific tumor types carrying the nanomedicine of interest. They can be loaded with different nanomaterials or drugs, including cytotoxic proteins, prodrugs, cytokines, oncolytic viruses, small molecule inhibitors, and immunotherapeutic payloads, and provide a promising platform to induce cancer cell death in a context-dependent manner

Fig. 4.

Nanotechnology-based strategies to modify or/and use macrophages for anticancer therapies. A-B The utilization of engineered particles termed as ‘backpack’ to regulate cellular phenotypes of macrophage(s) in-vivo. Backpacks are specialized particles that evade phagocytosis for several days and secrete out the cytokines that help guide the polarization of naïve macrophages to M1-macrophages, an antitumor phenotype. These M1 phenotypes are durable and substantially reduce the growth of cancer cells. C Macrophages can be utilized as a direct carrier of anticancer drugs or nanomedicines. D The membrane of macrophages is a promising tool to encapsulate nanomedicines. E CAR-Ms is a potential immunotherapeutic strategy to kill tumor cells. The specific CAR-Ms can be engineered using nanotherapeutic approaches to enhance their anticancer efficacies

Biological Ligands for Tumor Cell Targeting

Functionalization of nanosystems with biological ligands like proteins and polysaccharides, aptamers, and small molecules, which can selectively interact and bind to the overexpressed receptors present on the surface of cancer cells, has been extensively exploited to enhance the internalization and retention of drug-loaded nanocarriers, particularly by tumor cells [73].

Antibodies and Proteins Decorated Nanosystems

Antibodies with a dissociation constant in the nano to picomolar range have outstanding specificity and affinity for antigens specifically expressed on the surface of tumor cells, therefore, they are considered to be the best and have the longest history among biological ligands for receptor-specific targeting [201–203]. Recent studies explored the use of monoclonal, bispecific, and fragmental antibodies for relevant targeting purposes. A guided missile-like system for the precise release of payload can be created by functionalizing drug-carrying nanoparticles with monoclonal antibodies against a specific target present exclusively in cancer cells [202, 204, 205]. For example, a comparative study conducted on the anti-tumor efficacy of the antibody–drug conjugates (ADCs) with cetuximab, which is an anti-epidermal growth factor receptor (EGFR) antibody, guided avidin-nucleic-acid nano assemblies (ANANAS), reported that cetuximab guidance for ANANAS significantly enhanced the drug-antibody ratio as compared to ADCs [206]. Similarly, in two different studies on breast cancer and lung cancer models, anti-EGFR monoclonal antibodies have been explored to evaluate the efficacy of rapamycin-loaded polymeric poly(lactide-co-glycolide) nanoparticle and paclitaxel palmitate prodrug nanosystems (Cet-ppl-NPs), respectively [207, 208]. Authors reported a higher uptake of conjugated nanoparticles with significantly superior anti-proliferative capacity as compared to unconjugated nanoparticles on MCF-7 cells [208]. Similar results have been obtained in the lung cancer model, where (Cet-pcpl-NPs) have shown higher cellular uptake and cytotoxic profile in A549 cells [207]. It is noteworthy to mention that in this study, cetuximab has been used only as a guiding moiety while its independent anti-cancer potential remains unexplored. Recently, Oh et al. utilized the concept of protein corona shield by decorating the surface of camptothecin (CPT) loaded silica nanoparticles with human epidermal growth factor receptor 2 (HER2) targeted highly engineered proteins having a smaller size than antibodies known as affibodies (Afbs) [209, 210]. Authors highlighted that the resulting Afb-CPT-nanoparticles of 270 nm in size have enhanced blood circulation time with accelerated cellular uptake by higher HER2 expressing SK-BR-3 cell lines of breast cancer, with a 90% reduction in the tumor size after giving through the intravenous route during ex vivo examination. Moreover, HER2-directed monoclonal antibodies like Trastuzumab (Herceptin®) and Pertuzumab (Perjeta®) have been manufactured due to the higher expression of HER2 in gastric, ovarian, and breast cancer models [211]. For instance, Her-docetaxel (DTX)-nanoparticles have been successfully nano-formulated with improved camouflage and better cellular uptake properties by decorating Herceptin to the surface of DTX loaded nanoparticles for precise and controlled release of the drug in the vicinity of breast cancer cells [212].

Transferrin (Tf), an 80-kDa glycoprotein, is responsible for iron transport and has been studied for a long time to decorate nanoparticles for tumor cells targeting having a higher expression of transferrin receptor (TfR) [213, 214]. For instance, Tf-decorated oxaliplatin-loaded liposome, MBP-426, for tumor targeting has been entered in a phase I clinical trial (ClinicalTrials.gov Identifier: NCT00355888), having 39 patients with advanced metastatic tumors [215]. MBP-426 showed a favorable safety index with the recommendation of a 226 mg/m² dose for phase II. Collectively, all these outcomes of research studies and clinical trials advocated that the surface modifications of nanoparticles by using antibodies or affibodies through protein engineering could pave the way for nanoformulations to escape from the phagocytic system, resulting in minimized adsorption of serum proteins due to the protein corona effect [216]. However, a substantial hindrance is the formation of a "protein corona" when NPs enter the bloodstream. This corona can mask targeting ligands like antibodies, reducing targeting efficiency. For example, a fact that the formation of protein corona on anti-HER2 antibody-functionalized lipid nanocarriers led to a significant reduction in cellular uptake by HER2^+ve breast cancer cells, which resulted in compromised targeting capability, has been established by the study conducted by Marchal et al. [217]. In this study, lipid liquid nanocapsules (LLNCs) functionalized with a fluorescent anti-HER2 antibody (αHER2) have been developed by the authors to target HER2-overexpressing breast cancer cells (SKBR3 cell line). Detailed cellular uptake studies revealed that SKBR3 cells exhibited a multiple-fold higher uptake of LLNCs-αHER2 compared to human dermal fibroblasts (HDFa), indicating successful targeting. However, when the LLNCs-αHER2 were coated with a protein corona (LLNCs-αHER2-PC), there was a significant reduction in uptake by SKBR3 cells, suggesting that PC does not entirely prevent specific cell targeting; it substantially influences the efficiency of cellular uptake [217].

Nanosystems having Polysaccharide Moieties

Being an essential source of energy and informational biological macromolecules, polysaccharides are considered to be indispensable for living systems. Polysaccharide moieties have gained tremendous attention to be used as targeting ligands for the precise delivery of nanoparticles to tumor cells due to their specific interactions with certain cell-surface proteins, along with excellent biocompatibility [218]. Hyaluronic acid (HA), one of the key components of the extracellular matrix, has been used frequently as a polysaccharide-targeting moiety, which can selectively recognized by the endogenous lectins, like two hyaluronic acid receptors, called cluster determinant 44 (CD44) and receptor for hyaluronan-mediated motility (RHAMM), highly expressed on the plasma membrane of cancer cells [219, 220]. Notably, a study conducted by Choi et al. reported that HA can simultaneously serve as the hydrophilic backbone for nanoparticles and a targeting ligand. Additionally, this study also highlighted that HA is degraded by the overexpressed enzyme of malignant cells, hyaluronidase 1 (Hyal-1), which eventually enhances drug release within targeted tissue [221]. Another study conducted by Fang and colleagues highlighted that CD44-overexpressing 4T1 breast cancer cells showed significantly better cytotoxic response to docetaxel-loaded multifunctional hyaluronic acid nanoparticles as compared to free docetaxel [222]. However, their clinical translation has been impeded by several challenges. One significant hurdle is the variability in polysaccharide composition and structure, which can lead to inconsistent targeting efficiency and therapeutic outcomes [223]. Additionally, the penetration of these NPs gets hindered due to the presence of dense extracellular matrix in tumors, which further limits their access to cancer cells. Moreover, the potential for rapid clearance by the mononuclear phagocyte system reduces the circulation time of these NPs, thereby diminishing their therapeutic efficacy [224]. Addressing these challenges requires standardized polysaccharide sources, surface modifications to enhance tumor penetration, and strategies to evade immune clearance.

Aptamers as Biological Ligands

Aptamers, also known as chemically designed antibodies, are short, immunogenic, and extremely sensitive, single-stranded nucleic acid-based molecules [225], offering a variety of unique characteristics like high stability and being easily modifiable with minimal immunogenicity [226]. They are specifically designed to show highly specific binding with a wide array of targets, ranging from small molecules to entire cells [227]. Owing to these advantages, multiple aptamers have been considered as well-suited candidates for active targeting ligands against various biomarkers expressed on the surface of cancer cells, like mucin-1, EpCAM (epithelial cellular adhesion molecule), prostate-specific membrane antigen (PSMA), and nucleolin [228]. The study conducted on mice model having xenograft of HeLa cells by Duo et al. demonstrated the potential of aptamers in cell-specific targeting by using the AS-1411 G-enriched aptamer of DNA to direct CX-5461 loaded mesoporous silica nanoparticles to penetrate specifically to tumor cells through binding with nucleoli, a highly expressed protein on the tumor cell surface [229]. Similarly, the extracellular domain of PSMA, which is a prostate-specific biomarker, has been targeted in two different studies by decorating PLGA nanocarriers having docetaxel or cisplatin with the 2’-fluoropyrimidine RNA aptamer A10 for selective killing of prostate cancer cells [230–232]. Recently, GBI-10, a single-stranded DNA-aptamer loaded with dimeric camptothecin prodrug, has been used to specifically target pancreatic ductal adenocarcinoma (PDAC) due to the strong binding affinity against tenascin-C, an overexpressed protein in PDAC [233]. In this study, the authors reported higher drug concentration at the site of the tumor at all time points with enhanced antitumor proliferative capacity, along with an increase in survival times as compared to control groups. Another study on human colon cancer by Xie et al. found accelerated cellular uptake of docetaxel-loaded EpCAM aptamers by SW620 cells but not in EpCAM-negative Ramos cells, with the former showing higher cytotoxicity [234]. Furthermore, it has also been reported that siRNA has been successfully delivered in high EpCAM expressing MCF-7 and WERI-Rb1 tumor cells by using EpCAM decorated PEI nanosystem, resulting in significant EpCAM silencing with the decrease in tumor cell proliferation [235]. Despite the substantial scope, high precision, and favorable outcomes in targeting nanoparticles by these aptamers, the main limitation is their reliance on the known biomarkers that may not be universally present across all cancer types. This problem has been solved by the development of a tool called (systematic evolution of ligands by exponential enrichment) SELEX [236–238], which emerged as a powerful approach to address this limitation. By leveraging the cell-based SELEX tool, it is possible to design a universal aptamer that has the capability to selectively identify cancer cells even in the absence of established biomarkers [239]. For instance, a Cell-SELEX-based system has been used by researchers to design sgc8, a DNA-based aptamer against protein tyrosine kinase 7 to target human leukemic CCRF-CEM cell lines [240, 241]. Further in vivo investigations revealed that docetaxel-loaded Sgc8 aptamers have excellent drug loading capacity and a higher ability to target protein tyrosine kinase 7-specific cancer cells as compared to different formulations of docetaxel in a murine xenograft model [242].

Recent decades observed a significant use of biotin, folic acid (FA), phenylboronic acid (PBA), and ACUPA (S, S-2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid), as small molecules for tumor cell-targeted therapies due to various advantages like easy to handle with high stability and low manufacturing cost [243]. FA has a higher potential to specifically bind and kill tumor cells, having a higher expression of folate receptors as compared to normal cells [244, 245]. For instance, a study conducted by Lv et al. reported that tanshinone IIA-loaded silica nanoparticles, having FA released the drug in the close vicinity of the endothelial layer in the target tissue, resulting in the apoptosis of higher FA-expressing HeLa cells as compared to A549 cells with a lower FA expression profile [246]. Further in vivo examination of this nanoformulation on a murine tumor model having H22 cells exhibits greater antitumor efficiency. In similar studies on breast cancer and human nasopharyngeal epidermoid carcinoma cells, authors reported that doxorubicin-loaded nanoparticles with FA functionalization showed improved off-targeting with enhanced cellular uptake in higher FA-expressing KB and 4T1 cells [247]. Another study that revealed the use of small molecules in tumor cell targeting was conducted by Deshayes et al. They established that there is a strong binding affinity of PBA to tumor cells having a higher content of sialic acid. Taking this phenomenon into consideration, their group designed the PBA functionalized nanoparticles having a payload of metal-based chemo drug, dichloro-(1,2-diamino-cyclohexane)-platinum (II) (DACHPt). They found that B16F10 cancer cells have a higher accumulation of PBA-modified nanocarriers in vitro with excellent tumor inhibitin0067 and antimetastatic efficiency in the murine cancer model as compared to nanosystems without PBA decoration [248]. Furthermore, Biotin (vitamin B7) and ACUPA (urea-based substrate analog inhibitor of PSMA) have also been explored by different studies for modification of nanoparticles to achieve specific cell targeting [249, 250]. For instance, a study conducted on therapy-resistant adenocarcinoma cells by Patil et al. highlighted that dual agent loaded polyethylene glycol–poly lactic acid-co-glycolic acid nanoparticles having tariquidar along with paclitaxel showed enhanced anti-neoplastic efficacy when functionalized with biotin as compared to the non-functionalized system [251]. Similarly, the decoration of docetaxel-loaded nanoparticles with ACUPA exhibited a substantial cytotoxic profile against human prostate adenocarcinoma LNCaP cells, having a higher expression of PSMA [252]. Owing to this outcome, PLA-PEG—PLA nanoparticle, named BIND-014, having docetaxel and decoration of ACUPA on the surface, has been designed by Hrkach and colleagues and subjected to preclinical examination with a variety of animal models, including mice, monkeys, and rats [243]. Based on the favorable outcomes of these studies like excellent anti-tumor efficacy due to higher intratumoral concentration of docetaxel with improved safety index, the Phase I clinical trial having 52 individuals suffering from advanced tumors (ClinicalTrials.gov Identifier: NCT01300533), exhibited well toleration with negative toxicity index and a recommendation of 60 mg/m² dose for ongoing phase 2 trials on 42 patients having metastatic CRPC (ClinicalTrials.gov Identifier: NCT01812746) [253, 254].

Sub-Cellular Specific Tumor Targeting

The conventional cytosolic uptake mechanism of drugs may be adequate for molecules with high intracellular stability; however, this does not hold for biologically active compounds like peptide drug conjugates and therapeutic oligonucleotides [79, 255]. The highly crowded and acidic environment of the cytoplasm impedes their efficacy by facilitating enzymatic breakdown. Therefore, therapeutic molecules must navigate to specific organelles and subcellular structures to achieve maximum therapeutic outcomes with minimal off-targeting. Emerging nanotechnology, referred as third-generation nanomedicines, offers a promising strategy to protect the stability of these compounds with higher clinical effectiveness at lower doses by enabling precise delivery to subcellular organelles like mitochondria, nucleus, endoplasmic reticulum, lysosomes, and Golgi apparatus, within tumor cells [78, 80, 256]. Moreover, recently conducted preclinical and clinical studies highlighted that nanomedicine designed to target subcellular organelles has the capacity to counter multidrug resistance (MDR) by ensuring targeted intracellular delivery and avoiding endosomal entrapment [257, 258].

Mitochondrial-mediated targeting

Besides being considered a powerhouse of the eukaryotic cell [259], the mitochondria have also been considered a crucial center of pro- and anti-apoptotic proteins, thus regulating cell death pathways [260, 261]. Being the center of convergence for various lethal suicidal signaling cascades, mitochondria promote tumorigenesis, therapy resistance, and unresponsiveness to anti-growth signals with inhibition in autophagy and, therefore, have now been considered as a gateway to cancer [260, 262, 263]. Interestingly, cancer cells have an extraordinary ability to survive in highly proliferative hypoxic environments due to the metabolic reprogramming in mitochondria, which stabilizes hypoxia-inducible factor-1 and shifts the metabolism from oxidative to glycolytic [264]. Therefore, in recent years, mitochondria have been considered to be one of the promising targets for cancer therapeutics. Owing to the characteristic continual membrane potential of − 180 to − 200 mV, mitochondria become the selective organelle for lipophilic cations like rhodamine 123, triphenylphosphonium (TPP), and anthracyclines to accumulate [265]. For instance, Gong et al. developed mitochondrial targeting MitoCAT-g, having gold (CAT-g) and payload of TPP and cinnamaldehyde (CA) to generate mitochondrial oxidative stress, resulting in apoptosis and inhibiting tumor growth and proliferation in a murine model of hepatocellular carcinoma [266]. In another study, authors decorated paclitaxel-based liposomes with rhodamine 123 and found that this modification substantially enhances the cytotoxicity of paclitaxel by specifically targeting mitochondria as compared to liposomes alone [267]. Recently, MITO-Porter, a unique liposomal-based nano-drug delivery system, has been developed by Yamada and colleagues for mitochondrial-specific targeting [268, 269]. Since then, it has been used as a mitochondrial-targeted drug delivery vehicle for various chemotherapy drugs. For example, delivery of gentamicin or doxycycline in two separate studies gives very promising results in terms of anti-tumor response by suppressing mitochondrial membrane potential and ATP synthesis in cervical cancer cell lines and renal cell carcinoma, respectively [270–272]. Besides the above-mentioned designs and strategies, multiple other approaches, including Colloidal dequalinium vesicles, Szeto-Schiller (SS) and KLA peptides, and near-infrared dye (NIR), have also been explored for the decoration of nanoparticles to enhance the mitochondrial targeting [273–277].

Naosystems against Nucleus

The Nucleus, the largest and key organelle of the multicellular organization, holds the majority of genetic information required for cell replication and DNA transcription to regulate the genetic makeup [278, 279]. Additionally, it has also been considered the primary site of activity for multiple chemotherapy drugs, including doxorubicin, cisplatin, and camptothecin [76, 280]. Therefore, it is necessary to develop more crafted nanosystems capable of directly delivering these drugs to the nucleus, resulting in enhanced treatment efficacy with reduced off-targeting. However, the majority of the hydrodynamic nanosystems are too large to pass through the 9 nm diameter of nuclear pore complexes (NPCs) [281–283]. To address this barrier of permeability, researchers have developed nuclear-localization signal (NLS), like simian virus-derived SV40 [284], and Trans-activating transcriptional activator (TAT) peptides, to functionalize nanocarriers and promote their active transport into the nucleus [285–287]. It has been well documented that nanoparticles functionalized with NLS are efficiently transported across the NPC by the nuclear receptor karyopherin-α/ karyopherin-β1 [288]. A study conducted by Smith et al. demonstrated the successful nuclear import of leukemia-targeting chimeric antigen receptor (CAR) genes into the nuclei of T-cells with the help of nanoparticles decorated with NLS associated with microtubules [289]. Similarly, nucleus-based photodynamic therapy has shown effective results in a recent study conducted in vitro and in vivo by using NLS functionalized exosomes having chimeric peptides with alkyl chains [289]. Another nuclear receptor exploited for nucleus-based targeting is importin-β, which interacts with 49 to 57 (RKKRRQRRR) highly basic residues of TAT and mediates the localization of proteins in the nucleus [287]. For example, it has been recently reported that irradiation of TAT peptide-functionalized nanoparticles having a payload of doxorubicin and photosensitizer (Ce6) at 660 nm efficiently triggers cell death in cancer cells by disrupting the nuclear membrane in MDA-MB-231 cells. Furthermore, in vivo studies also advocated the use of this nanosystem by significantly inhibiting metastasis in tumor-bearing mice [290]. Besides TAT and NLS-mediated strategies, dexamethasone, a synthetic glucocorticoid, has also been used recently to improve nuclear targeting and internalization in conjugation with micelles loaded with doxorubicin [291].

Endoplasmic Reticulum-mediated targeting

Endoplasmic Reticulum (ER), an important organelle, plays a key role in calcium homeostasis and serves as a functional site for numerous enzymes [292]. Being the central hub for key pathways and signaling cascades, ER is considered to be requisite for cell fate and survival, while any dysfunction in these processes results in ER stress [293, 294], which can be used to initiate cancer cell death through apoptosis by promoting the cleavage of caspase 8 or by activating anti-tumor immunity by enhancing the expression of immunogenic proteins on the surface of cancer cells [295, 296]. Like, the targeting of ER in human lymphoma cells with the small molecule called hexaminolevulinate leads to the induction of apoptosis by damaging the ER calcium pump protein and cleavage of caspase 8 [297]. Moreover, it has also been reported that Lys–Asp–Glu–Leu (KDEL) motif and KKXX, an ER retrieval signal that can be identified by the KDEL receptor, can be used to achieve ER-targeted strategies. For instance, gold nano constructs in conjugation with the KDEL motif have been synthesized by Wang et al. to effectively deliver the nano formulation to the ER, resulting in the escape from lysosomal degradation with enhanced drug efficacy [298]. Recently, ER-targeted therapies have been revolutionized by the use of Pardaxin (FAL) peptides for the functionalization of indocyanine green conjugated nanospheres of gold, resulting in the FAL-ICG-HAuNS and liposomes having hemoglobin (FAL-Hb-lipo). These nano systems, regarded as ER-guided missiles, trigger enhanced release of cytotoxic chemokines and eventually lead to immunogenic cell death [299]. In addition to the above-mentioned robust strategies of nanotechnology for ER targeting to increase the anti-tumor efficacy of chemotherapy drugs, other biological moieties, like the use of vitamins, p-Toluene sulfonamide (PTSA), poly (aspartic acid) (PAsp), and CDPP-SO_3, have also been employed [300–302].

Nanosystems against the Golgi Complex

The Golgi complex is another important cell organelle and protein destination, having a unique structure of cisternae [303, 304]. Owing to the key role in the trafficking of proteins and lipids, and the formation of lysosomes [305], any abnormalities in the functioning or glycosylation of Golgi apparatus (GA) are meticulously related to the proliferation and metastasis of tumors [306], hence it has now been emerged as a potential target for nanomedicines in modern oncology [307–309]. For instance, chondroitin sulfate and retinoic acid nanoparticles have been developed with the payload of paclitaxel, demonstrating the significant reduction in the process of angiogenesis, proliferation, and migration in vitro and the dramatic reduction of tumor growth and metastasis in vivo by disrupting the Golgi structure [310]. Recently, Golgi-targeted lipid-based nanoparticles having a payload of doxorubicin and retinoic acid have been designed by Luo et al. Authors reported that the resulting nanoformulation has been rapidly taken up by the SMMC-7221 cells and showed higher tumor penetration with significant accumulation in the Golgi complex, resulting in the reduced expression of extracellular matrix and apoptosis [311]. Another important moiety designed for Golgi-specific targeting of cancer cells through interaction with the sulfhydryl receptors present in the lumen of the Golgi is the six-cysteine peptide (C6) [312]. Li and colleagues performed a study on MCF-7 and A549 cells, in which self-assembled nanoparticles were decorated with C₆ at the N-terminal and a C- C-terminal having a hexapeptide (F4KY), while in between them, there is a tetrapeptide (RVRR). This resulting C6RVRRF4KY nanosystem causes significant mechanical damage to cancerous Golgi, resulting in reduced cytokine secretion with a collapsed structure of the Golgi apparatus. Notably, repeated stimulation of tumor cells by these nanoparticles did not exhibit any acquired form of drug resistance [313]. Aside from these ligands and strategies, a series of novel biological moieties like aminoquinolines, derivatives of cysteine, and phenylsulfonamide have been explored to date to target the Golgi network [314, 315].

Lysosomal Disruption by Nanoparticles

The lysosomal system, a hub for many hydrolytic enzymes, not only serves the role in cell digestion but also plays a significant role in the regulation of cell death mechanisms by governing the release of hydrolytic enzymes in the cell cytoplasm [316, 317]. Therefore, the capacity of lysosomes to induce cell death has been exploited by introducing the lysosome-targeted nano carriers for cancer therapy, and the traditional view of lysosomes as mere "waste disposal" units has evolved [318, 319]. For instance, the endocytic pathway has been used by Chen and colleagues to deliver nano photosensitizers, which can sense the acidic environment of lysosomes and get activated to trigger the oxidative stress in them, thus leading to cancer cell death through pyroptosis in orthotopic tumor implantation [320]. Specifically, lysosomal enzymes, cathepsins, have a role in the invasion of cancer cells by regulating the angiogenesis and apoptosis [321], therefore, cathepsin B surface sequence Arg-Arg has been used to decorate the lysosomal targeted Au-ZnO nanoparticle for the induction of apoptotic cell death through reactive oxygen species formation in HepG2 cell lines [322]. Similarly, utilizing the acidic environment of lysosomes, Du et al. developed the pH-sensitive, poly (ethylene glycol)-b-poly(2,4,6-trimethoxybenzylidene-1,1,1-tris(hydroxymethyl)ethane methacrylate)-b-poly (acrylic acid) (PEG-PTTMA-PAA) nanoparticle of doxycycline having an acetyl bond between PTTMA and PEG-PAA. These acetal bonds get hydrolyzed by the acidic pH as soon as nanoparticles get internalized by lysosomes, resulting in the efficient release of the drug [323]. Another strategy for lysosomal targeting is the delivery of nitric oxide (NO) and reactive oxygen species donors by designing morpholine-decorated lysosomal-specific nanoparticles. For example, nanoparticles having carbon doping with titanium oxide (Lyso-Ru-NO@FA@TiO2), have been developed by Xiang et al. These nanoparticles have a payload of folic acid and ruthenium nitrosyl donor with morpholine modifications, resulting in lysosomal accumulation and enhanced anti-tumor potential as compared to non-targeted nano formulation [324]. In a recently published report on hepatocellular carcinoma, Wang and colleagues developed a curcumin-loaded low toxic nano system having integration of morpholine and galactose functionalization. These nanoparticles have dual targeting capacity due to the overexpression of ASPGR in hepatoma cells and lysosomal specificity for morpholine moiety, therefore, the resulting nano formulation exhibited enhanced endocytosis, better biocompatibility with an excellent hepatic targeting profile as compared to free curcumin or liposomes having only galactose modification [325].

Smart Tumor Targeting Strategies: Advancing the Drug Delivery

Despite noteworthy and remarkable developments in tumor-specific delivery of nanoparticles, some inevitable hurdles in terms of size, surface charge, and nonspecific interactions of nanodrugs with the blood components are still there [326]. For example, nanoparticles must have an optimal size of 100–150 nm with neutral or anionic character to achieve tumor accumulation by the EPR effect and extended circulation time [327, 328]. However, on the other hand, smaller size nanoparticles with cationic charge are well known to show the “proton sponge effect”, resulting in deep penetration inside the tumor through the nuclear pore complex by escaping the endosomal degradation [329, 330]. Although nanoparticles functionalized with targeted ligands are capable of accumulating more efficiently in tumor cells as compared to traditional nanomedicines, the inexplicit interaction between serum proteins and these ligands and leakage of drugs during circulation still hold true [331, 332]. Owing to these hurdles, a stable nanosystem with definite physicochemical properties is still quixotic. Therefore, to address these issues, researchers have exploited either endogenous or exogenous stimuli present in the tumor microenvironment to produce stimuli-responsive nanoparticles [333]. These nanocarriers can shield their ligands from the external environment and have the capacity to unmask them on their surface once they reach the site of action in response to changes in either internal stimuli like pH, enzymes, hypoxia response, or external stimuli, including ultrasound, and magnetic fields (Fig. 5) [333]. Furthermore, these stimuli-responsive nanoparticles can also trigger a change in size and charge, resulting in the controlled drug release and deep penetration of the tumor with productive and enhanced anti-tumor efficacy [334, 335]. Therefore, to define important and adequate methods of efficient tumor targeting by dynamically combining all the above-mentioned strategies, we contemplate “smart” strategies of nanotherapeutics for oncology to boost the performance of nanoformulations and maximize the treatment outcome.

Fig. 5.

Schematic illustration of smart tumor targeting approaches with nanoparticles. A Various types of nanoparticles with specific modifications. B Representative method to utilize stimulus-specific nanoparticles as a carrier of anticancer nanomedicines to cancer cells. The nanoparticles are designed in a precise way so that they can deliver the loaded drug or biological agent under the influence or once activated with a specific stimulus such as heat, ultrasound, light, magnetic field, pH, redox status, and enzyme activity. C The representative mechanism for pH-sensitive nanoparticles encapsulating drug(s) or/and siRNA. The pH-sensitive nanoparticles are a promising strategy to deliver specific drugs or drug-like molecules. These particles release drugs once internalized by cancer cells, as they were designed to release encapsulated material in a pH-dependent manner. The low pH of cancer cells is a key phenomenon to utilize these particles. These types of particles act as a potential nanomedicine strategy to deliver multiple types (s) of drugs or small interfering RNAs such as siRNA, miRNA, epigenetic modulators, immune-activating drugs or chemokines, or antibodies, which will help to induce cell death in cancer cells

Nanosystems Responsive to pH Change

The tumor internalization and penetration of nanoparticles have been improved by the design and development of pH-responsive nanoformulations. These nano drugs exploit the acidic tumor microenvironment (6.5–6.9), and the pH gradient observed in sub-cellular compartments of cancer cells due to the rapid production of lactic acid [336, 337]. For instance, pH stimuli-responsive iCluster nanoparticles have been developed by Li et al. to achieve deep penetration of BxPC3 pancreatic tumors [83]. These nanocarriers have an EPR-favorable diameter of 100 nm at the physiological pH range. However, when it reaches the site of action, the acidic tumor microenvironment triggers the size shrinkage to an approximate diameter of 5 nm, which makes it the best fit for deep penetration of pancreatic tumors, resulting in improved treatment outcomes with maximum therapeutic efficacy [83]. Furthermore, these iClusters are not only capable of deep tumor internalization but also inhibit metastasis by translocating into the lymph nodes [338]. Subsequently, Wang and colleagues developed pH-responsive inorganic platform-based nanoparticles by enveloping the coordination complex of 4-phenylimidazole and Zn²⁺ with CaCO₃, to transform the immunosuppressive tumor microenvironment [339]. In another study, a pH-responsive nanocarrier bearing a negative charge and zeta potential of -22.9 mV with multistage targeting potential has been developed by Mo et al. [340]. Once this nanosystem reaches the tumor site, the acidic microenvironment and subcellular compartments trigger the change in zeta potential to + 6.3, and it keeps fluctuating to the value of + 22.5Mv. This pH-responsive charge conversion facilitates the internalization with mitochondrial targeting by electrostatic bonding between negatively charged membranes [340]. A recent study conducted by Liu et al. screened the library of “proton transistor” nano detergents (pTNTs) to design and develop ultra pH-sensitive nanocarriers with higher specificities for tumor tissues [341]. Authors reported that at higher pH, these pTNTs have the capacity to self-assemble to form neutral nanoparticles with no membrane lysis property. However, when pH changes to the acidic range, these pTNTs achieve cationic properties, resulting in tumor cell membrane solubilization. Notably, the precision of this material can be advocated by the fact that the membrane-cleaving material can sense the pH change of 0.1 [341]. However, several limitations hinder their clinical efficacy. One primary challenge is the heterogeneity of tumor pH. While tumors generally exhibit a more acidic environment compared to normal tissues, the pH can vary significantly within different regions of the same tumor and among different tumor types. This variability can lead to inconsistent drug release and reduced therapeutic effectiveness [342].

Enzyme-Responsive Release of Nanoparticles

Enzymes play an important role in numerous pathways and processes [343]. It has been well established that enzymes like Matrix metalloproteinase (MMPs) and hyaluronidase (HAase) are affiliation with various cancers, and they are exclusively overexpressed in the tumor microenvironment with rare or no expression in normal tissue [344–346]. Thus, enzyme responsiveness has been exploited by researchers back-to-back to design various drug delivery systems [347–349] to effectively transport the payload by enhancing the bioavailability with a reduced toxicity profile [350]. For instance, MMP2 overexpressing tumor microenvironment has been exploited by Naz and colleagues to develop nanoparticles functionalized with PVA peptides (PPCs) [351]. Authors demonstrated that the PEG shell had been removed from PPCs, resulting in their transformation into β-sheets in response to the cleavage of MMP2, leading to the better retention of drugs with improved efficacy in immune checkpoint inhibition [351]. Similarly, Liu et al. developed CD44-targeted nanoparticles having size shrinkage capability in response to higher accumulation of HAase in breast cancer. Authors reported significant tumor inhibition and lung metastasis in the tumor-bearing murine model [345]. Moreover, to augment enhanced tumor internalization, γ-glutamyl transpeptidase (GGT)-responsive nanoparticle having charge conversion capacity has been developed by Zhou et al. [352]. Cleavage of an overexpressed GGT enzyme on the cell membrane and luminal surface of tumor cells having higher metabolic activity converts neutral nanoparticles into cationic ones, resulting in deep tumor penetration and enhanced anti-cancer properties due to caveolae-mediated endocytosis in the BxPC3 orthotropic pancreatic cancer murine model [352]. In a recent study, one of the lysosomal proteases, known as Cathepsin B (CTSB), has been exploited by Wang et al. to develop TLR7/8 agonist-conjugated nanovaccine (TNV) having enzyme-responsive linker Gly–Phe–Leu–Gly (GFLG). Following the administration, antigen-presenting cells phagocytosed the TNV, resulting in the exposure of GFLG to cathepsin B, which ultimately leads to the free release of imidazoquinoline to sensitize the TLR7/8, located on the endosomal inner membrane [353]. Besides the above-mentioned enzymes, numerous other enzymes like protease K, β-glucuronidase, glutathione (GSH), and Alkaline phosphatases have been exploited for multi-stage tumor targeting strategies [354–357].

Oxygen-Responsive Nanoparticles

Rapid cell growth and intense oxygen need are two major characteristics of tumor cells [358], resulting in the abnormal development of blood vessels and scarcity of oxygen, especially in deeper tumor regions [359]. This hypoxic tumor microenvironment not only hampers drug delivery but the efficiency of other therapies like photodynamic, sonodynamic, and chemodynamic therapies, which rely on oxygen to produce ROS for tumor cell destruction, also get affected [360, 361]. Advances in material science have introduced hypoxia-responsive components, such as nitroaromatics and azobenzene linkers, that remain stable in non-monocytic conditions, however, any change in the oxygen concentration activates them, resulting in the targeted drug release and triggering therapeutic reactions within the tumor [362]. For example, a unique albumin-based hypoxia-responsive nanosystem has been designed by Yang et al. [363]. They reported that nanoparticles having an initial size of 100–150 nm are stable under normoxic conditions. However, when the system reaches the hypoxic environment of tumor cells, the reductases present there cleave the azobenzene group, resulting in the disassembly of one nanosystem into multiple nanoparticles of size 10 nm with therapeutic potential [363]. Recently, an imaging probe having a derivative of nitroimidazole, ultrasmall iron oxide (UIO) nanoparticle, and fluorescence dyes has been developed by Zhou and colleagues [364]. Larger and irreversible formation of nanoparticle assemblies has been documented in this study with the enhanced signal of magnetic resonance imaging, resulting in the clear display of hypoxia content in tumor regions within the whole murine model [364]. Furthermore, hypoxia-responsive elements have also been exploited for delivery and an on-trigger release of chemotherapy drugs or immune adjuvants in the tumor microenvironment [365, 366]. For instance, Hao et al. synthesized paclitaxel-loaded hypoxia-responsive nanoparticles with high drug content with azobenzene as a cleavable linker [367]. They demonstrated that severe hypoxic conditions in the core tumor region resulted in the cleavage of azobenzene and the release of cytotoxic drugs in the cancer cells. Furthermore, hypoxia-responsive photo trigger, TAP, has been developed by Lin et al. to enhance the efficacy of chemotherapeutic agents while sparing normal cells [368], an approach that aligns with other studies, which established the selective targeting of hypoxic tumor environments by photodynamic therapy. However, in some cases, hypoxia is insufficient to elicit a rapid therapeutic response. Owing to this, not only functionality of oxygen-responsive NPs designed to alleviate hypoxia become limited, but also hampers the effectiveness of oxygen-dependent therapies, such as photodynamic therapy (PDT). Moreover, the heterogeneous distribution of oxygen within tumor tissues leads to inconsistent NP activation and therapeutic outcomes [369]. Therefore, certain specific methods to intensify hypoxia within tumors, such as photodynamic [367, 368] and vascular-disrupting agents [370], have been explored. Given that the conventional use of photodynamic or chemotherapy against solid tumors having both normoxic and hypoxic regions struggles to show promising outcomes. Therefore, to target both superficial and deeply embedded tumor cells simultaneously, Xiong et al. introduced hypoxia-sensitive chemical agents and photodynamic therapy, resulting in enhanced treatment efficacy across varying oxygenation zones [371]. Collectively, tumor-induced hypoxia is known to cause hindrance in therapeutic outcomes, and cancer patients with a greater extent of hypoxia usually show poor survival and prognosis [372]. Nanomedicine not only delivers oxygen-loaded materials in the tumor core but also exploits the hypoxic characteristics of cancer cells to develop smart tumor-targeting strategies by turning the hypoxic environment from the therapy suppressor to the therapy enhancer.

Challenges in Successful Clinical Translation of Nanomedicines

Nanomedicines have observed and enjoyed impressive progress over the last 3 decades, with the enormous increase in the global market showing continuous signs of growth. At the same time, concern about their successful translation in clinical settings has arisen due to the disparity between the huge number of research publications on nanomedicines and their limited translation into clinics, as shown in Table 3, with the recent approval of Onivyde (liposomal irinotecan) and Vyxeos (liposomal daunorubicin plus cytarabine) [373]. However, at present, it would be too early and might be unjust to underemphasize the potential and ongoing development of nanomedicines for cancer therapeutics. Therefore, to enhance the probability of nanomedicine projects running in academic institutions to gain clinical attention and translation, we promote and endorse a more aggressive attitude to encourage nanoscience projects to have the capacity for clinical translation with product development.

Table 3.

Clinical trials for actively targeted Cancer nano drugs in different therapeutic modalities (2010–2022). Data was collected from the clinicaltrials.gov database

Therapeutic modality	Proprietary name	Nanocarrier	Target	Title	Conditions	NCT number	Phase	Outcome
Chemotherapy	ABI-009	Albumin-bound rapamycin	mTOR	Nab-sirolimus in Recurrent High-Grade Glioma and Newly Diagnosed Glioblastoma	High-Grade Recurrent Glioma and Newly Diagnosed Glioblastoma	03463265	II	Completed
	CPC634	Docetaxel micellar	β-tubulin	Efficacy Study of CPC634 (CriPec® Docetaxel) in Platinum-Resistant Ovarian Cancer (CINOVA)	Ovarian cancer	03742713	II	Completed
	EndoTAG-1	Liposomal paclitaxel	Tumor endothelial	A Trial Evaluating the Efficacy and Safety of EndoTAG®-1 in Combination with Paclitaxel and Gemcitabine Compared with Paclitaxel and Gemcitabine as First-line Therapy in Patients with Visceral Metastatic Triple-negative Breast Cancer	Triple-Negative Breast Cancer	03002103	II	Suspended
				EndoTAG-1 + GEM vs GEM in Patients with Locally Advanced/Metastatic Pancreatic Adenocarcinoma Failed on FOLFIRINOX	Locally Advanced Pancreatic Cancer Metastatic Pancreas Cancer Pancreatic Adenocarcinoma	03126435	III	Completed
	MM-302	Liposomal doxorubicin hydrochloride	HER2	MM-302 Plus Trastuzumab vs. Chemotherapy of Physician's Choice Plus Trastuzumab in HER2-Positive Locally Advanced/Metastatic Breast Cancer Patients	Breast Cancer HER2 Positive Breast Cancer	02213744	III	Terminated
	CRLX101	NPs consisted of Camptothecin-cyclodextrin conjugate	topoisomerase-1	Pilot Trial of CRLX101 in Treatment of Patients with Advanced or Metastatic Stomach, Gastroesophageal, or Esophageal Cancer That Cannot be Removed by Surgery	Stomach, gastroesophageal, esophageal cancer	01612546	II	Completed
	NK105	Paclitaxel micelle	β-tubulin	A Phase III Study of NK105 in Patients with Breast Cancer	Breast Cancer Nos Metastatic Recurrent	01644890	III	Completed
	NKTR-102	Irinotecan PEG conjugate	Topoisomerase-I	The BEACON Study (Breast Cancer Outcomes With NKTR-102)	Locally Recurrent Breast Cancer Metastatic Breast Cancer	01492101	III	Completed
Radiotherapy sensitizer	NBTXR3	Hafnium oxide NPs	Radio enhancer	NBTXR3 Crystalline Nanoparticles and Radiation Therapy in Treating Randomized Patients in Two Arms with Soft Tissue Sarcoma of the Extremity and Trunk Wall	Adult Soft Tissue Sarcoma	02379845	III	Completed
				NBTXR3 Crystalline Nanoparticles and Radiation Therapy in Treating Patients with Soft Tissue Sarcoma of the Extremity	Adult Soft Tissue Sarcoma of extremity	01433068	I	Completed
				NBTXR3 and Radiation Therapy in Treating Patients with Locally Advanced SCC of the Oral Cavity or Oropharynx	Head and Neck Cancer	01946867	N/A	Active, not recruiting
				NBTXR3 Activated by Radiation Therapy for the Treatment of Locally Advanced or Borderline-Resectable Pancreatic Cancer	Pancreatic Ductal Adenocarcinoma	04484909	I	Recruiting
				NBTXR3, Radiation Therapy, and Pembrolizumab for the Treatment of Recurrent or Metastatic Head and Neck Squamous Cell Cancer	Metastatic/Recurrent Head and Neck Squamous Cell Carcinoma	04862455	II	Recruiting
	NVX-108	DDFP liquid emulsion combined with radiation therapy	Radiation Sensitizer	The Effects of NVX-108 as a Radiation Sensitizer in Glioblastoma	Glioblastoma Multiforme	02189109	I	Completed
	AGuIX®	A polysiloxane matrix with gadolinium cyclic chelates covalently grafted on the inorganic matrix	Radiation Therapy	Radiosensitization of Multiple Brain Metastases Using AGuIX Gadolinium Based Nanoparticles	Brain Metastases	02820454	I	Completed
				Stereotactic Brain-directed Radiation with or Without Aguix Gadolinium-Based Nanoparticles in Brain Metastases	Brain Metastases Brain Tumor Brain Cancer	04899908	II	Recruiting
				Nano-SMART: Nanoparticles with MR Guided SBRT in Centrally Located Lung Tumors and Pancreatic Cancer	Pancreatic Adenocarcinoma Non-small Cell Lung Cancer	04789486	I & II	Recruiting
				Radiotherapy of Multiple Brain Metastases Using AGuIX®	Brain Metastases, Adult	03818386	II	Active, not recruiting
Hyperthermia	NanoTherm	Aminosilane-coated Fe3O4 superparamagnetic nanoparticles	Generate heat	Study of Focal Ablation of the Prostate with NanoTherm® Therapy System for Intermediate-Risk Prostate Cancer	Prostate Cancer	05010759	N/A	Terminated
				NanoTherm In Adjuvant Therapy of Glioblastoma Multiforme	Glioblastoma Multiforme	06271421	N/A	Recruiting
	AuroLase	Silica-gold nano shells coated with (poly)ethylene glycol (PEG)	Generate heat upon illumination	Efficacy Study of AuroLase Therapy in Subjects with Primary and/or Metastatic Lung Tumors	Primary or Metastatic Lung Tumors	01679470	N/A	Terminated
				Pilot Study of AuroLase(tm) Therapy in Refractory and/or Recurrent Tumors of the Head and Neck	Head and Neck Cancer	00848042	N/A	Completed
Immunotherapy	Personalized mRNA cancer vaccine	Personalized mRNA cancer vaccine against neoantigens	Tumor-associated antigens (TAAs) and neoantigens	Messenger RNA (mRNA)-Based, Personalized Cancer Vaccine Against Neoantigens Expressed by the Autologous Cancer	Colon Cancer Gastrointestinal Cancer Hepatocellular Cancer Melanoma	03480152	I & II	Terminated
				Clinical Study of Personalized mRNA Vaccine Encoding Neoantigen in Patients with Advanced Esophageal Cancer and Non-small Cell Lung Cancer	Esophageal Cancer Non-Small Cell Lung Cancer	03908671	N/A	Recruiting
				Study of Personalized Tumor Vaccines and a PD-L1 Blocker in Patients with Surgically Resected Pancreatic Adenocarcinoma	Pancreatic Cancer	06156267	I	Not yet recruiting
	CV9202	mRNA cancer vaccine in combination with anti-PD-L1	Melanoma-associated antigen-C1 & C2	Phase 1/2 Study of Combination Immunotherapy and Messenger Ribonucleic Acid (mRNA) Vaccine in Subjects With NSCLC	Metastatic Non-small Cell Lung Cancer	03164772	I & II	Completed
	DPX-Survivac	Liposomal survivin-based synthetic peptide antigens	Survivin	DPX-Survivac and Checkpoint Inhibitor in DLBCL	Adult Diffuse Large Cell Lymphoma Adult Refractory Diffuse Large B-Cell Lymphoma Recurrent	03349450	II	Completed
				DPX-Survivac and Pembrolizumab with and without Intermittent Low-Dose Cyclophosphamide, in Subjects with Relapsed/Refractory Diffuse Large B-Cell Lymphoma	Refractory Diffuse Large B-cell Lymphoma Relapsed Diffuse Large B-cell Lymphoma	04920617	II	Recruiting
				Phase 1b Study of a Cancer Vaccine to Treat Patients with Advanced Stage Ovarian, Fallopian, or Peritoneal Cancer	Epithelial Ovarian Cancer Fallopian Tube Cancer Peritoneal Cancer	03332576	I	Completed
	PNT2258	Liposomal DNA interference oligonucleotides PNT100	Regulatory region upstream of the BCL2 gene	Study of PNT2258 for Treatment of Relapsed or Refractory Non-Hodgkin's Lymphoma	Lymphoma, Non-Hodgkin's	01733238	II	Completed
				A Study of PNT2258 in Patients with Advanced Solid Tumors	Cancer Lymphoma Melanoma Prostate Cancer	01191775		Completed
	Vaccination with mRNA transfected DC	Vaccination with mRNA transfected DC	Specific antigens with known epitopes	Peptide-pulsed vs. RNA-transfected Dendritic Cell Vaccines in Melanoma Patients	Melanoma Stage III or IV	00243529	I & II	Completed
				Safe Study of Dendritic Cell (DC) Based Therapy Targeting Tumor Stem Cells in Glioblastoma	Brain Tumor Glioblastoma	00846456	I & II	Completed
	Tecemotide	Liposomal MUC1 antigen	MUC1	Tecemotide (L-BLP25) in Prostate Cancer	Prostate Cancer	01496131	II	Completed
				Tecemotide (L-BLP25) in Rectal Cancer	Rectal Cancer	01507103	II	Completed
				Phase 2b Randomized Controlled Study of Tecemotide (L-BLP25) for Immunotherapy of NSCLC (Non-Small Cell Lung Cancer)	Carcinoma, Non-Small-Cell Lung Lung Neoplasms	00157209	II	Completed
				L-BLP25 in Patients with Colorectal Carcinoma After Curative Resection of Hepatic Metastases	Colon Carcinoma Rectum Carcinoma	01462513	II	Completed
Nucleic acid nano drug	SGT-53	Liposomal p53 plasmid	Transferrin receptor	Safety Study of Infusion of SGT-53 to Treat Solid Tumors	Neoplasm	00470613	I	Completed
	Atu027	Liposomal protein kinase N3 siRNA	Protein kinase N3	Study With Atu027 in Patients with Advanced Solid Cancer	Advanced Solid Tumors	00938574	I	Completed
				Atu027 Plus Gemcitabine in Advanced or Metastatic Pancreatic Cancer (Atu027-I-02)	Carcinoma, Pancreatic Ductal	01808638	I & II	Completed
	pbi-shRNA STMN1 LP	shRNA against stathmin 1 using lipid NPs	Stathmin 1	Phase I Intratumoral Pbi-shRNA STMN1 LP in Advanced and/or Metastatic Cancer	Advanced Cancer Metastatic Cancer Solid Tumors	01505153	I	Completed
	siRNA-EPHA2-DOPC	siRNA against EPHA2 using liposomes	EPHA2 receptor tyrosine kinase	EphA2 siRNA in Treating Patients with Advanced or Recurrent Solid Tumors	Advanced Malignant Solid Neoplasm	01591356	I	Active, not recruiting
	ALN-VSP02	siRNAs against KSP and VEGFA using lipid NPs	Kinesin spindle protein and Vascular Endothelial Growth Factor A	Multi-center, Open Label, Extension Study of ALN-VSP02 in Cancer Patients Who Have Responded to ALN-VSP02 Treatment	Solid Tumors	01158079	I	Completed
				Dose Escalation Trial to Evaluate the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Intravenous ALN-VSP02 In Patients with Advanced Solid Tumors with Liver Involvement	Solid Tumors	00882180	I	Completed

Real-World Feasibility, Cost-Effectiveness & Market Potential

One of the foremost thing to be considered before the development of any nanoformulation is the practical and commercial feasibility [374]. In this aspect, it is necessary to achieve improved treatment outcomes, an unsophisticated delivery system with a few components, manufacturing production should be cost-effective with high standards of quality control, and a maximum number of benefited patients. It is important to develop cost-effective and scalable NP as their inherent complexity renders them difficult to produce on a large scale and provide suitable storage conditions when at the pharmaceutical level [66]. Furthermore, the manufacturing process of multi-targeting nano systems often requires complex path, resulting in less efficient production and inconsistent results. In contrast, the use of microemulsion techniques, microfluidic systems, nanoprecipitation, and self-assembly is easily scalable, cost-efficient, and high yield output [375]. In a study, the microfluidic system was utilized to successfully and efficiently deliver the paclitaxel-loaded lipid nanoparticles as chemotherapy drugs. The use of such a system enabled an appropriate control over the size of the particle in the form of liposomes measuring less than 200 nm with high stability, uniformity, and low polydispersity index. Several drugs have been tested with this approach, enabling better encapsulation capability and sustained release of the drugs with a flow rate of 1 ml/min and a 1:4 flow rate ratio. Therefore, offering a strategy for lipid-based nanocarrier formulation that is both reproducible and scalable and, in turn, offers improved and safe chemotherapeutics [376]. Furthermore, the idea of automation has substantially reduced the dependency on manual operators and increased the efficiency and cost-effectiveness of the product. For instance, replacing traditional batch synthesis with continuous flow reactors has provided more consistent NP production [377, 378]. Treatment benefits to patients can be achieved by enhancing the therapeutic efficiency, with a less toxic profile and number of doses required as compared to the comparator product. A product with more unique and clearer benefits can attract investors and commercial industries to step in with the justification of the premium price. Here, the mindset of patients and clinicians is important because it is necessary to know whether they are ready to adopt the treatment regime of active pharmaceutical ingredients, which require less frequent dosing but come with multiple times increases in the treatment cost as compared to their parental cheap tablet freely available in the market. Collectively, it is less favorable to recover huge amounts of dollars spent on the development of nanomedicines until unmatched therapeutic benefits with better efficacy in long-term treatment regimes have been shown by the resulting product. It is important to consider the ease of scalable production initially when designing the nanovehicle [379]. Therefore, the lower complexity of nanodrug preparation helps in achieving the desired product with less cost invested, good reproducibility, and large-scale production.

Reproducibility, Screening, and Precision Synthesis

Another important aspect in the development of nanomedicine is the reproducible synthesis, evaluation, and screening to address the discrepancies between in vitro and in vivo outcomes of pre-clinical studies [105, 380]. It is necessary to ensure the improvement in the in vitro examination of nanoparticles by introducing organoids [381] and cell-to-cell interactions through co-culture techniques before performing animal studies. With the ongoing research focused on nanoparticles, it is of utmost importance to ascertain the most favorable physicochemical parameters for developing a successful therapeutic nano formulation [382, 383]. In this regard, significant literature is available, which is focused on the factors that display successful immune system evasion, selective tumor targeting, and internalization potential with controlled and sustained release of chemotherapy drugs [231, 328, 384, 385]. Despite this knowledge, we still lag in the systematic screening of nanoparticles, and the lack of quick and robust libraries with distinct features for stable and replicable synthesis imposes a major challenge. The conventional technique of bulk nanoparticle synthesis with a higher polydispersity index has grown old and has been overtaken by microfluidic technologies. The nanoparticles synthesized with this new technique have a narrow size distribution, which is not offered by the conventional type of synthesis. Also, it is easier to fine-tune the chemical and physical properties of the product [386–388]. The high-speed synthesis, self-assembly characteristics, and greater batch reproducibility, in turn, offer additional likelihood for the scientists waiting for fast and reliable nanoparticles to be produced [389, 390]. At the same time, another important aspect is the screening of their biocompatibility before they can be tested on animals [105]. In vitro testing of nanosystems can be done to study their interaction with cells and their components. The reluctance with conventional techniques using multi-well plates is that they fail to offer knowledge about the complex interplay occurring in biological tissues and lack control over fluid flow [391]. Also, it is very difficult to predict how nanomedicines will behave in crossing the physiological barrier once inside the animal system [373]. The efforts to develop biomimetic tools such as the tumor/organ on a chip method can help ease this drawback of conventional in-vitro assay, due to their capacity to use the dynamic microfluidic platform to recapitulate the architecture and microenvironment of cancer cells, they also serve as an operational and sophisticated adjunct for conventional animal models. Additionally, they also allow to conduct the temporospatial modulation of biophysical and biochemical parameters, like the stiffness of extracellular matrix, composition of cell and interstitial fluid, more precisely. In particular, advancements made in these platforms empower the analyses of convoluted crosstalk among primary human-derived cell populations under physiologically admissible and highly controllable conditions [392–394]. Owing to these advantages, recent years have seen a significant increase in the improvement of modeling tumor physiology through the development of microfluidic devices with variable complexity, to understand the effects of the architectural aspect of tissue on the transport and internalization of nanoparticles. For instance, Agarwal et al. synthesized a three-dimensional (3D) tumor architecture by encapsulating cancer cells in the shells of microcapsules having the core of collagen and a thick periphery [395]. After reaching the size of microtumors, these cancerous capsules have been combined with stromal cells having human umbilical vein endothelial cells (HUVECs) and human adipose-derived stem cells (hADSCs), resulting in the 3D spatial arrangement of tumor cells with HUVECs designed in capillary-like structures. Additionally, authors reported that they found 100 times more doxorubicin resistance in the cells under 3D arrangement as compared to the 2D system. Notably, this resistance of vascularized 3D systems has been overcome by the treatment of nanosytems decorated with photosensitizer indocyanine green and doxorubicin. Furthermore, a remarkable feature of this system is the utilization of fundamentals of self-organization, which empowers the use of polydimethylsiloxane-based microfluidic system to generate wavering vascular networks in 3D space. Despite significant advancements, there is a limitation in mirroring the real tissue in terms of direct contact between the tumor compartment and applied solutes. On the other side, in a real physiological system, the entry of solutes is either by a convective flow where fluids leave the capillary vasculature through a lymphatic drainage system, or via a diffusion process, depending on the interstitial pressure. In case of a vasculature-mimicking multi-compartment model, an endothelial cell layer is applied, which is very different from the one where a perfusion channel is separated from the compartment of the tumor cells using an aperture to create a physical barrier [396]. Likewise, an easy-to-use, multichannel, and microfluidic system to ascertain the impact of perfusion is the use of a three-compartment device where peripheral channels lie on the sides of a central compartment. These peripheral channels are, in turn, separated from the central compartment with the help of pillars, which serve the function of a discontinuous endothelium or to support the growth of endothelial cells. In another application, the extracellular matrix-embedded tumor cells are loaded in the central compartment, and drugs are then permeated via one of the peripheral channels. The pressure difference between the two peripheral channels is maintained such that the perfusion of drugs into the tumor compartment can be made either through convective flow or a diffusion process. This type of system has been used to validate the working model for diffusion-mediated transport of engineered cell-targeting proteins [397]. Another variant of this system comprises weirs/barriers for capturing spheroids. Here, spheroids comprising tumor cells and fibroblasts are incorporated, which helps in assessing the tumor-cell specificity of retargeted adenovirus [398]. This application directly aids in nanomedicine research. However, not denying the fact that the in-vivo performance of these nanoparticles is mandatory to be studied in animal models. It was also seen that there is still a discrepancy in the efficacy obtained in the pre-clinical trials when compared to the clinical trials. The major contributing factor to these differences is the lack of tumor models that can mimic human tumors [399]. Despite the available tumor models, including the subcutaneous cell-line-based and orthotropic xenografts, patient-derived xenografts (PDXs), and genetically engineered mouse models, there is no such model that can prove successful in reproducing all aspects of human malignancy [73, 400]. In this regard, a model of human tumor metastasis may prove a valuable approach for the evaluation of penetration potential, enhanced permeability, and retention of nanoparticles, and also for targeting the metastatic tissues compared to primary tissues. Translating nanotherapeutics can be improved to variable degrees by developing animal models that closely mimic the heterogeneity, histological anatomy, and other metastatic features of human tumors. The idea of developing PDXs with high fidelity, GEM models with aggressive metastasis, and humanized mouse models may prove successful in this regard. [401–403]. For example, a study conducted by Sahli et al. on two different kinds of TNBC-PDX models, one derived from a patient with paclitaxel resistance, and the other from the patient without previous exposure to chemotherapy, established the successful delivery of EGFP-mRNA payload by using PLGA-PEG based nanoparticle system [404]. Additionally, the recruitment of chimpanzees, monkeys, and other non-human primates has also been recommended by recent studies for late preclinical studies before initiating clinical trials [405].

Translation into Clinical Settings

Designing clinical trials is the critical challenge that needs to be addressed to overcome the current limitations and ensure their successful completion. In this aspect, designing the basic research and clinical trials on nanomedicines should be done to investigate the deep drug action mechanisms, increase in survival rate, toxicity profile, and adverse events with other clinically relevant factors, rather than just a comparison of efficacies between the new nano product and the standard existing therapies, which is not enough to get funding support from pharmaceutical industries [406]. Furthermore, the high degree of variation among cancer patients due to the unwanted interactions between nanoparticles and serum proteins is another major hurdle in successful clinical trials. To address this issue, merely tapering the inclusion criteria is not an option because it hampers the statistical significance and reliability of the patient outcomes. Since nanomedicines have been designed to target specific oncogenic pathways associated with the progression and advancement of the disease, it is feasible to implement the biomarkers as the fortunetellers of therapeutic outcomes, along with the solution for high inter-patient variability. Additionally, dissimilar to conventional therapies, having toxicity issues due to the active pharmaceutical ingredient, nanomedicines have even more biosafety concerns from the nanocarrier itself. Therefore, it is necessary to do a rigorous examination of the toxicity profile and biosafety risk assessment by studying the interaction of nanocarriers with the tissues and serum components before the clinical trials to encourage a greater number of subjects [374, 391].

Artificial Intelligence and Machine Learning: The Next Frontier in Cancer Nanomedicine

The ongoing advancements in biomedicine and nanotechnology have led to cancer therapeutics becoming progressively personalized and data-intensive as well. The scientific community at the global level is making efforts, right at the very beginning, to optimize the personalized combination therapy to achieve better efficacy, efficiency, and the lowest possible toxicity of the administered doses. The aim of achieving these goals brings into action some very important aspects of artificial intelligence that can help in the analysis of multi-source and multi-dimensional data, namely, machine learning (ML), deep learning (DL), and artificial neuronal networks (ANN) [84, 407]. For example, a study conducted by Verma et al. established a novel strategy for the design and optimization of electromagnetic plasmonic nanostructures by using a highly advanced and automated method of ANN [408]. Notably, the ANN model rapidly and accurately predicted optical responses, outperforming the traditional method of finite element in computation time. Moreover, the robustness of this process has been validated by high prediction accuracy with low mean squared error. This approach offers a fast, reliable tool for designing plasmonic devices, advancing sensing and photonic technologies. While designing complex nanoparticles, it is important to understand the features of nanocarriers and their respective nanomedicines, along with the understanding of correctly evaluating their cyto and hemocompatibility, chemical and physical properties, possible interaction of the loaded drugs, and pharmacokinetics [409]. Proper understanding of the above-mentioned properties is of utmost importance and can be achieved with the use of AI. Although the incorporation of AI with nanomedicines is still in its early stages but the general prediction regime of the nanodrugs to be loaded on nanocarriers can be achieved. AI can also help in deciding the biological mediators/ cell membranes, the drug encapsulation efficiency, drug release kinetics, and optimizing the drug formulation for enhanced transportation and targeting of the nanomedicines. AI has also helped in predicting the synergy between the patient's response to treatment and the drugs prescribed. The patient response evolves during the treatment in a time and dose-dependent manner; therefore, this prediction will help personalize the treatment regimen. The current strategy to design therapeutics at a fixed dose might fail hence, the predictive AI has given a boost to the clinicians in utilizing a large-scale data-driven approach, genetics-based, and broad omics profile-based approach to design patient-specific treatment of the given population [410]. A recent study utilized this predictive AI feature to boost the efficacy of a combination therapy designed for breast cancer cells by combining three nano-diamond modified drugs and one unmodified drug, also, simultaneously they optimized the biosafety in normal cells [411]. Also, this study was able to successfully rule out the use of randomly designed nanomedicine combinations that are more toxic than being efficacious. The AI-optimized nano-drug combinations were found to be more efficacious, have a better biosafety profile when compared with randomly combined or individual drugs. Another versatile feature of AI is the ability to address population heterogeneity and non-biological processes without the requirement of cellular signaling pathways and omics data. Additionally, prior optimization of drug ratios will help achieve enhanced drug efficacy rather than pose an additive effect, for example, if we combine mitoxantrone, doxorubicin, and bleomycin, as these three are very similar. Although this field is fast growing, it is still facing many challenges, one of them being the limited availability of large datasets for designing in-silico models and training of AI. In this regard, the use of nanosensors, maintaining the multi-omics profile of patients by sequencing technology, availability of the treatment responses, diagnostic and image monitoring, can be utilized and should be made available to the data scientists for developing AI-based functions. There is a need to develop collaboration between the medical researchers, scientists, clinicians, and AI data scientists to critically evaluate the collected data in building up the AI models and bringing them into algorithms.

Conclusions and Future Outlook

The research field of nanomedicine continues to grow, with researchers focusing on targeting new disease indications and gaining momentum in the clinical arena, but this was never an easy-going setting. However, the path to clinical success remains challenging. This review was therefore conceptualized to dig deeper and create awareness regarding nanosystem therapeutics right from the very beginning of design formulation till the end user to enhance patient benefit. Key aspects like the use of AI and ML for the designing and synthesis of nanoparticles, understanding the heterogenous tumor nature, identification of EPR markers, ensuring nanoparticle reproducibility, screening, and evaluation, optimizing interactions between nano-bio systems, and transportation of nanoformulations to tumor microenvironment will play a crucial role in enhancing nanomedicine performance, translation, and clinical applications.

The therapeutic inefficiency of nanomedicines is one of the major reasons for therapy failure in clinical trial studies. This issue is particularly prominent in phase-II and phase-III clinical settings, where the inability to overcome physiological barriers is a critical hurdle [66]. EPR-mediated accumulation of nanoparticles in tumors has been proposed as a solution, though its effectiveness is limited. This EPR-mediated accumulation of nanoparticles is mainly derived from the murine xenografts. However, its translation into solid tumors of human origin showed inconsistent results, with varying outcomes between patients due to large variations in the deposition of drugs and their delivery system at the tumor site, also, the journey that is followed by the delivery system is highly extensive before it can reach the tumor site [412, 413]. This journey is initiated with the drug formulation being administered in the system via blood, where it has to evade the opsonization by plasma proteins, avoid liver and spleen uptake, and bypass the detection from the reticulo-endothelial system. The delivery system needs to be present for a sufficient time in the system so that it can leak into the tumor interstitium via the truncated tumor endothelium [414]. The past few years have seen research more inclined to design nanotherapeutics to pass these obstacles; however, at present, more attention is focused on achieving the end-stage penetration into the tumor core [415, 416]. This successful penetration of NPs largely affects their results and efficacy as observed in a highly controlled model of the tumor with minimum variability. In contrast, clinical trials with humans were subjected to significant variability as a consequence of gene alterations or epigenetic changes, and this variability is observed in either of the cases: different types of tumors and different tumors of the same type [377, 417, 418]. The incorporation of artificial intelligence could further aid in designing strategies that generate nanoparticle libraries tailored to specific patient needs [407]. Personalized pro-drug and nanocarrier designs are expected to maximize the therapeutic potential of nanomedicines in individual patients. The use of tumor-on-a-chip and in vitro 3D models to mimic the complex tumor environment of human physiology is emerging as a promising solution to study the process of tumor penetration by nanodrugs in a more comprehensive manner [419–422].

The progression of research based on cellular and physiological mechanisms has led to an increase in the studies on inter-organelle interactions and their collaborative efforts in maintaining cellular homeostasis. This understanding facilitates research on the effect of multi-organelle collaboration in designing tumor therapeutics. As maintaining cellular homeostasis is not an individual task but a collective effort, this synchrony between various organelles is essential for cells to survive in unfavorable conditions and adapt to the changing environment [423]. In this regard, the interaction between the endoplasmic reticulum and lysosomes helps in regulating cholesterol metabolism to support lipid homeostasis [424]. Different signaling pathways, membrane contact sites, and complex communication pathways help in coordination among organelles [425]. When these interactions are dysregulated, it results in disease onset, for example, the overexpression of IP3R (a mitochondrion-associated membrane protein), which mediates the cross-talk between endoplasmic reticulum and mitochondria, is implicated in glioblastoma and breast cancer [426]. Hence, with the advancements in AI, nanoparticle designs that have the capability of multi-organelle targeting have shown considerable success for cancer therapies. In a study conducted by Fu et al., a ferroptosis nanocarrier was designed in the form of an inhalable biomineralized liposome, which was co-loaded with dihydroartemisinin-DHA along with a pH-responsive calcium phosphate. This system resulted in an increased Ca²⁺ burst, the trigger being the calcium phosphate shell degradation facilitated by DHA-induced SERCA inhibition. This further resulted in disrupting mitochondrial functioning and enhanced ROS accumulation, enhancing lung cancer ferroptosis therapy [427]. A different study reported a new therapy, namely-COMET-cellular mitochondria-ER therapy, for the treatment of multidrug-resistant TNBC [428]. Their approach was based on the delivery of anti-MFN peptides by utilizing the drug-loaded NPs. Here, in this process, the main aim was to interfere with the apoptosis-related process by altering the mitochondrial integrity (changes in its morphology, networking, and reduction in mitochondrial footprints). At present, the most efficient strategy is thought to be the eradication of the ER-Golgi system of the tumor for inhibiting tumor growth [429]. The efficacy of tumor immunotherapy was found to be enhanced by a regulatory mechanism where interaction between MFN-2 and SERCA-2 proteins present at the mitochondrion-ER site helps to modulate the function and metabolism of cytotoxic T-lymphocytes, thereby promoting MFN-2 expression levels [430]. The same approach was utilized by Guo et al., where cancer CMs were used to envelope polymeric micelles loaded with 1G3-Cu and Toy (GCT@CM NPs), which helped in enhanced apoptosis of tumor cells and ICD by taking advantage of the IG3-Cu induced oxidative stress and mitochondrial damage [431]. As ER and GA can play an important role in tumor eradication, the recent years have seen increased focus of researchers on nanodrugs to precisely target these two organelles. In this instance, a group of researchers designed two synthetic peptides, EVMSe and E3C16E6, having strong affinity for ER and GA, respectively [432]. The peptides were GSH responsive, enabled self-sorting and organelle-specific targeting of GA and ER by disrupting their function and inducing tumor cell death, thereby providing a novel drug delivery strategy. Although there is considerable advancement in the design of nanodrugs that simultaneously target two organelles but at the same time, if more organelles are added, the challenge increases. As the number of organelles increases, there is difficulty in the preparation of therapeutically efficient designs. However, there is still debate about increasing the number of organelles and overcoming the complexity associated with the therapeutic efficiency. This area needs to be explored and is still not completely addressed to overcome the drawbacks.

Another strategy to enhance cellular internalization and achieve an effective treatment regime, the functionalization of nanocarriers with specific targeting ligands can be a good option. In this regard, it is important to understand that the ligand should neither be immune-responsive nor toxic to the system. The formation of biomolecular corona is another obstacle in this regard. This corona is formed because of the nanoparticle meeting biological fluids, including blood or interstitial fluids, simultaneously adsorbing biomolecules on the surface of nanoparticles. Thereby, preventing the binding of ligand-bound nanoparticles to their respective receptors located on tumor cells [433]. Another problem regarding biomolecular corona is that it may be immunogenic in nature. Therefore, there is always a question of nanoparticles getting cleared from the system before reaching their targets [433–435]. This can be prevented by organelle-specific targeting of nanomedicines [436], the differences in various organelle's signature, their size, charges, and structure can be exploited, but the differences in types of cells and their physiological properties make it indeed difficult to design a single strategy for a specific organelle in various cell types [437].

The internalization of nanoparticles is another complex issue, as different cell types use different endocytic pathways (e.g., clathrin-mediated, caveolae-mediated, or phagocytosis). [438]. A successful strategy to overcome this problem is through the surface modification of NPs, including the charge, shape, and size, which helps in the sorting and uptake of NPs by the endocytic pathways and their uptake by specific organelles [438]. Additionally, ensuring that nanoparticles are not trapped in endosomes or lysosomes is crucial for effective drug delivery. Innovative approaches, such as triphenylphosphonium derivative (TPT) nanoparticles, hyaluronic acid-coated particles, and polymeric nanovehicles, show promise in improving nanoparticle circulation time and tumor accumulation, enhancing therapeutic efficacy [334, 439–443]. One particularly promising strategy is the development of "smart" nanocarriers with targeted drug delivery systems, which could be applied in self-administered therapies. For instance, antibody-based therapies such as Humira (adalimumab) against TNF-α, which can be self-administered subcutaneously at home, highlight the potential of less toxic, cost-efficient, and easy application treatments [444]. Although such approaches have been underexplored, they hold significant potential for improving patient convenience, reducing treatment costs, and minimizing toxicity. Overall, the research should prioritize practical approaches that can be easily applied in real-world settings. By focusing on these strategies and turning innovations into actual therapies, we can fully realize the potential of nanomedicines and make them more widely available to help more patients.

Authors’ contributions

K.M.A. and J.A.S. conceptualized and designed the review. K.M.A. wrote the initial draft of the manuscript. K.M.A., G.S., A.P.S., and J.A.S. critically revised and/or edited the manuscript. All authors have read and approved the final version of the manuscript.

Funding

This work and the authors are, in part, supported by grants from the U.S. Department of Defense (DOD) through the Prostate Cancer Research Program under Award No. (DOD) W81XWH-21–1-0640 (JAS).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent to participate

Not applicable.

Competing interests

The authors declare no competing interests.