KRAS mutation-driven enhanced macropinocytosis: using albumin as a trojan horse for delivering nanomedicine

ABSTRACT

Kirsten Rat Sarcoma Virus Oncogene Homolog (KRAS), the most mutated oncogene, drives tumoral progression by leveraging macropinocytosis. This nutrient uptake pathway is responsible for promoting the relentless growth of KRAS-mutant tumoral cells while also unlocking a new frontier for the exploration of innovative therapeutic approaches. Leveraging KRAS-driven macropinocytosis, researchers are instigating advanced nanoparticulate drug delivery systems, including albumin nanoparticles and DNA- and RNA-loaded lipid nanoparticles. We emphasize the emerging strategies to exploit KRAS-macropinocytosis-albumin crosstalk presenting a promising avenue for the delivery of albumin nanoparticles in cancer therapy.

1. Introduction

Cancer continues to be a leading cause of death worldwide, with estimates suggesting nearly 2 million new cases will be diagnosed in the United States in 2024 [1,2]. American Cancer Society reports that almost 48% of all cancer cases in men majorly include prostate, lung, and colorectal cancers, while about 51% of total cases in women account for breast, lung, and colorectal cancers [3]. According to the American Cancer Society, by the year 2050, this number is predicted to increase to 35 million [4]. Cancer is a multifactorial disease; protein overexpression and mutations are crucial in cancer development, thus hindering the normal functioning of proteins [5].

Approximately one-third of the cancers arise due to mutations occurring in RAS proteins. RAS genes were named “Rat sarcoma viral” as a reference to how they were first discovered three decades ago [6]. RAS proteins are crucial in controlling the cascade of cell processes like cell proliferation and division. RAS protein functions as a binary signaling switch that toggles between “on/active” and “off/inactive” states. In its inactive state, the Ras protein binds to guanosine diphosphate (GDP), while in its active state, it binds to guanosine triphosphate (GTP). The regulation of Ras proteins involves a precise cycle between these two forms: the active GTP-bound state and the inactive GDP-bound state. In the mutated RAS proteins, intrinsic GTPase activity is compromised, leading to continuous activation of RAS proteins. As shown in (Figure 1), the uninterrupted activation of the RAS protein causes uncontrolled growth of cells, functioning as an oncoprotein, thus driving oncogenic signaling pathways [7].

Figure 1.

The EGFR family of receptor tyrosine kinases is crucial in signaling pathways for various cancers. When a ligand binds to EGFR, it undergoes autophosphorylation and recruits SH2 domain-containing proteins, linking the receptor to the SOS1 protein. SOS1 then helps convert KRAS-GDP to KRAS-GTP, which triggers downstream signaling pathways related to cell proliferation, survival, and cancer development. In the inactive state of KRAS-GDP, there is no signal transduction, leading to a lack of proliferative signaling pathways.

The RAS oncogene family comprises more than 150 distinct members but primarily includes the three widely known isoforms that are found in human tumors: the Harvey rat sarcoma viral oncogenes (HRAS), the Kirsten rat sarcoma viral oncogenes (KRAS) and the neuroblastoma rat sarcoma viral oncogenes (NRAS) [8]. These three RAS proteins are closely related in mammalian cells and promote oncogenesis when mutationally activated. Out of all the point mutations occurring in RAS proteins, the key oncogenic mutations in the RAS isoforms usually concentrate at three hotspots – the codons 12, 13, and 61 [9]. KRAS is the most mutated RAS oncogene, making it the predominant isoform among all the mutationally activated RAS proteins, followed by NRAS and HRAS [10,11]. Various human tumors selectively depict mutations in RAS isoforms, where KRAS mutation is widely observed in the adenocarcinoma of the pancreas, colon, rectal, and lung. Amongst all the RAS mutations, NRAS is found to be the second most frequent mutation, which is often found in hematological malignancies like chronic myelomonocytic leukemia and acute myeloid leukemia, having mutational rates of nearly 14%. NRAS mutations have also been found prominently in melanoma, thyroid carcinoma, and laryngeal carcinoma, with 18.6%, 8.1%, and 9.7% mutational frequencies. Meanwhile, HRAS mutations are rare but relatively higher in the mouth, salivary gland, and vulva carcinomas [11]. Researchers have been exploring protein domain mutations, like those in protein kinase domains, P53, PI3K, EGFR, RAS, BRAF, and PTEN protein domains, determining it crucial for understanding cancer prognosis, severity, and treatment response [5]. Various efficacious drugs like idelalisib, VO-OHpic, SF1670, gefitinib, erlotinib, prima-1, APR-246, sotorasib, and adagrasib represent a major breakthrough in targeting specific protein domains which were once thought to be undruggable. Notably, sotorasib and adagrasib have been FDA-approved for targeting mutated KRAS proteins [5].

2. Unraveling from KRAS mutation to medicine

From discovering the KRAS gene to developing targeted therapies today, scientists have faced a long and quite challenging path. KRAS is the most widely mutated oncogene and has been considered an elusive and unreachable target for decades. Initial therapy for KRAS-mutant cancer patients was suggested to be chemotherapy and immunotherapy. However, the recent breakthroughs in targeting KRAS using inhibitors like sotorasib and adagrasib paved the way for developing promising therapeutic strategies and overcoming drug resistance.

2.1. Decoding KRAS mutations and its role in oncogenesis

KRAS is a key signal transducer regulating cell proliferation, differentiation, endocytosis, migration, and survival, primarily through pathways including PI3K/AKT pathway for metabolism and/or survival, RAF/MEK/ERK pathway for regulating cell proliferation, and RalGEF/TBK1/NFκB pathway for cytokine production [12,13]. According to The Cancer Genome Atlas (TCGA), KRAS mutations are highly prevalent in various cancers. KRAS mutations are key genetic drivers in carcinomas and adenocarcinomas, including non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and pancreatic ductal adenocarcinoma (PDAC) [10,14]. Researchers suggest KRAS mutations are the hallmark feature in more than 80% of PDAC patients [15,16]. In patients with colorectal cancer, the KRAS mutation frequency is nearly 40%, while approximately 20% of the NSCLC patients exhibit KRAS mutations. Although less frequent, KRAS mutations are also found in testicular germ cell cancer, choloangiocarcinoma, breast cancer, cervical squamous cell carcinoma, and uterine endometrial carcinoma [12,16,17]. KRAS mutations can be classified into various subtypes based on their nature and location including KRAS G12V, KRAS G12R, KRAS G12D, KRAS G12C, KRAS G13D, and KRAS G12A mutations or KRAS wild type where each of these variants represents a different amino acid substitution at codons 12 or 13 of the KRAS gene with nearly 84% of the KRAS mutations occurring at G12 position [10,18]. The majority of KRAS mutations contain 21 missense mutations, with the most frequent ones being G12D (29.19%), G12V (22.97%), and G12C (13.43%). Literature suggests that KRAS mutations are highly prevalent in PDAC, with G12D being the most common subtype of KRAS mutation. However, in patients with NSCLC, amongst all KRAS mutations, KRAS G12C is the most common mutant subtype, followed by G12V and G12D [17]. KRAS mutations affect the cancer prognosis, leading to poor outcomes, further emphasizing targeted and effective approaches to heighten the effectiveness of the offered treatment [12].

2.2. Therapeutic tactics for KRAS-driven cancers

Due to the high incidence of KRAS mutations in different cancers, targeting the mutant KRAS protein directly and indirectly is an attractive strategy. The standard of care for patients exhibiting KRAS mutations is chemotherapy [10,17]. While RAS proteins have long been considered “undruggable,” growing evidence indicates that developing direct inhibitors of RAS proteins may be achievable [6]. Recent studies have explored using direct and indirect inhibitors that target mutant KRAS protein, immunotherapy, combination therapy, and targeted mutant KRAS protein degradation, including PROTACs, vaccines against KRAS mutant cancers, and small interfering RNA therapies [10,17]. After more than three decades of intensive research aimed at discovering RAS-directed therapeutics, there have been two therapies that the Food and Drug Administration approved against KRAS-driven cancers. Over the past four years, the FDA has provided accelerated approval for Krazati (adagrasib) from Mirati Therapeutics, Inc., when used alongside cetuximab for adult patients with KRAS G12C-mutated locally advanced or metastatic CRC and NSCLC [19,20]. Additionally, the FDA has granted accelerated approval for sotorasib, the first drug designed to inhibit KRAS, previously known as the AMG510. This groundbreaking treatment is specifically for adults suffering from KRAS G12C-mutated locally advanced or metastatic NSCLC, offering hope for a new therapeutic option [21]. Sotorasib and adagrasib are both covalent, selective inhibitors targeting the KRAS G12C mutation – a substitution of glycine with cysteine at codon 12. These inhibitors bind irreversibly to the cysteine residue that locks the switch II pocket in an inactive GDP-bound KRAS G12C protein, thus inhibiting the downstream oncogenic signaling, and leading to apoptosis [22,23]. Despite their promising clinical outcomes, resistance to KRAS G12C inhibitors frequently arises, majorly by adapting to KRAS bypass signaling pathway or secondary KRAS mutations [24,25]. Furthermore, their activity majorly relies on the KRAS G12C mutations, which excludes patients with other KRAS mutations including G12D or G12V [26]. To address the mutation-specific limitations and development of resistance to sotorasib and adagrasib, pan-KRAS inhibitors (BI-2865, PF-07934040) and degraders (ACBI3) are being explored to target a broad spectrum of KRAS variants, with pan-KRAS inhibitors, such as PF-07934040, LY4066434, already entering early-phase clinical trials [27–29].

3. Metabolic programming of KRAS mutated cancers

RAS protein is involved in key pathways that supply the KRAS mutant cancers with metabolic and nutrient sources to fuel their proliferation [13]. These mutations increase the uptake of glucose, glutamine, and fatty acids to sustain cell proliferation through the altered biosynthetic pathways. The three key findings highlight the metabolic reprogramming in KRAS-driven cancers – Firstly, by varying the origin of the tissue and the type of tumor, KRAS mutations significantly impact metabolic adaptation. Secondly, KRAS mutant cells exchange cytokines, growth factors, and metabolites in the microenvironment to improve their survival rates in nutrient-poor environments. Thirdly, metabolic reprogramming is further made possible by co-occurring genetic changes, which promote the development and survival of KRAS mutant cells [30]. One of the scavenging pathways for the survival and growth of KRAS mutant cancer cells is via macropinocytosis and autophagy. Cancer cells with mutant KRAS status have a greater tendency for macropinocytosis activities than those with the nonselective endocytic route [18]. The proliferation of cancer cells for growth and survival is highly dependent on macropinocytosis, which plays a crucial role by serving as a route to provide essential nutrients [31]. Enhanced macropinocytosis coupled with lysosomal degradation is an important mechanism for scavenging nutrients to support cell growth [30,32].

3.1. Exploring cellular gateways

The scavenging or cellular uptake mechanisms act as gatekeepers on the plasma membrane that govern the internalization of substances, including nutrients, therapeutic agents, and antigens, to regulate the appropriate cell functioning and survival. Small molecules like ions and amino acids usually traverse the membrane via the protein pumps/channels. Whereas macromolecules are engulfed into the cells via the process of endocytosis that involves the formation of membrane-bound vesicles. Two key mechanisms involved in endocytosis are phagocytosis, which refers to “cell eating” of larger molecules, and pinocytosis, which refers to “cell drinking” or the uptake of solutes and fluids. Cell internalization via pinocytosis can further be categorized by four distinctive uptake mechanisms, namely, caveolae-dependent endocytosis, clathrin-dependent endocytosis, caveolae- and clathrin-independent endocytosis, micropinocytosis and macropinocytosis [33]. Macropinocytosis is an endocytic pathway independent of clathrin- and caveolin-mediated process that allows for large-scale and nonselective uptake of solutes in different cell types, with immune cells, fibroblasts, and cancer cells. Additionally, it causes the cancer cells to proliferate under stress rapidly and are susceptible to targeted therapies [34].

3.2. Overdriving macropinocytosis in KRAS-mutant cancer

The environmental factors and biological function of the cell influence the macropinocytic activity, which is found in nearly all cell types but is significantly pronounced in KRAS mutant cancer cells. Unlike all the endocytic pathways that involve the binding of cargo molecules to the receptors that are present on the cell surface, macropinocytosis is an endocytic pathway that is an actin-dependent process that leads to the formation of large membrane ruffles and, hence, is not saturated on the cell surface. These membranes tend to fold back and fuse with the plasma membrane to form large intracellular vesicles called macropinosomes. The diameters of these macropinosomes are larger (0.2 to 5 µm) in size compared to other endocytic vesicles. It plays a crucial role in internalizing large volumes of extracellular fluids and performs various cellular processes, including the uptake of nutrients, entry of pathogens, and cell signaling [35–37]. Studies confirm that KRAS-transformed (oncogenic) cells substantially show an increase in the membrane ruffling characteristic of macropinocytosis to fulfil their metabolic dependency and help maintain their growth and survival [35,38]. A group of researchers indicated that oncogenic RAS enhanced macropinocytosis in pancreatic cancer cells, potentially leading to enhanced cellular uptake of exosomes where approximately 9-fold enhanced macropinocytosis was observed in Panc-1 cells than in BxPC-3 cells [39]. Elevated levels of macropinocytosis in KRAS-mutant cancer cells are well-documented, but it has been observed that exact levels largely vary. The inconsistency in data is likely due to differences in experimental conditions concerning time points and the presence/absence of serum, types of methods used for analysis (macroscopy vs FACS), and use of different models (cell lines vs tissues). Additionally, various studies prefer using immortalized wild-type KRAS cell lines like NIH3T3, BxPC-3, and MCF-7 as controls that do not truly represent normal human cells [18]. Activated KRAS mutations in lung, pancreatic, and colorectal cancers actively scavenge lipids, glutamine, and especially albumin to meet their metabolic demands [35]. Thus, leveraging this natural phenomenon of providing the KRAS mutant cells with albumin as metabolic support may prove to be an excellent therapeutic strategy for delivering and targeting KRAS-driven cancers [13].

4. Delivery of albumin nanoparticles to enhance macropinocytosis in KRAS-mutant cancers

Amongst all the widely used therapeutic biomolecules used as carriers for targeted drug delivery, human serum albumin (HSA), bovine serum albumin (BSA), and ovalbumin are commonly utilized to develop albumin-based nanoparticles [40]. HSA is the most abundant plasma protein and accounts for nearly half of the total protein content in healthy individuals [41,42]. HSA (66.5 kDa polypeptide, 585 amino acid sequence) is a highly water-soluble polypeptide that helps transport nutrients and various biomolecules like hormones, lipids, and amino acids [43]. Albumin from bovine serum (69.3 kDa polypeptide, 583 amino acid sequence) is like HSA and is inexpensive, easy to purify, and has high loading capability having the ability to bind to both hydrophilic and hydrophobic drugs, making it a very versatile carrier [44,45]. After HSA and BSA, ovalbumin (42–47 kDa polypeptide, 386 amino acid sequence), usually found in egg white, is used as albumin drug conjugates for various anticancer agents [44]. Albumin proves to be an ideal material for creating nanosized drug delivery systems due to its inherent properties like offering enhanced surface area, biocompatibility, biodegradability, non-immunogenicity, and extended half-life [40]. The ability of albumin to bind to endogenous and exogenous large biomolecules makes it an excellent carrier for drugs [46]. Literature has revealed that abundantly available plasma proteins have been extensively internalized into the tumor, and albumin has been widely explored in various anticancer drugs like paclitaxel, gemcitabine, and doxorubicin [43,47,48]. Accumulation of albumin in the tumor tissue fundamentally occurs through the following two mechanisms: Enhanced Permeation and Retention, also known as the EPR effect, and receptor-mediated internalization. Unlike the normal tissues that do not allow the transport of biomolecules via the vascular endothelium that are tightly connected, solid tumors inherently have leaky vasculature and poor lymphatic drainage that easily allow the macromolecules, such as albumin, to accumulate into the tumor tissue. This is known as the EPR effect, which allows passive targeting of tumor cells [43,49]. Nanoparticles that have been conjugated with albumin act as dysopsonin and can shield circulating albumin nanoparticles from recognition and removal by the mononuclear phagocytic system [42]. Due to the heightened metabolic needs of KRAS-mutated cells, enhanced macropinocytic uptake of albumin to support metabolic needs has become one of the major scavenging pathways [35]. A promising therapeutic approach is to exploit the inherent mechanism of albumin uptake by KRAS-driven cells. Sutton et al. demonstrated that enhanced uptake and retention of albumin are driven by the elevated macropinocytic activity associated with KRAS mutations in lung and pancreatic cancer cell lines [13]. Another study showed preferential uptake of cross-linked albumin nanoparticles by oncogenic KRAS-mutant breast cancer cells via macropinocytosis, emphasizing a potential strategy for exploiting RAS-driven metabolic vulnerabilities for targeted drug delivery [35]. An in vivo study in genetically engineered mouse models of KRAS-mutant lung and pancreatic cancer highlighted that strong co-localization of labeled albumin and paclitaxel within tumor cells. This indicated that the uptake is dependent on KRAS activity and silencing KRAS expression hindered drug accumulation of nab-paclitaxel [50,51].

4.1. Mimicking nutrient-like uptake for albumin nanoparticles

Albumin-loaded nanoparticles fuel macropinocytosis-driven internalization in KRAS-mutant cells and improve the delivery of drugs to the tumor site [50]. The human body has several albumin drug binding sites that include GP18, GP30, GP60, Secreted Protein Acidic and Rich in Cysteine, which is also known as SPARC, neonatal Fc receptor for IgG (FcRn), cubilin, and megalin [46]. FcRn is a receptor on the cell membranes, including vascular endothelium, intestinal epithelium, and kidneys. However, FcRn has a low affinity for native albumin and is majorly involved in maintaining the albumin concentration and protecting it from degradation. Conversely, the presence of glycoprotein receptor (GP) 60 on the vascular endothelium helps in the efficient transcytosis of albumin by rescuing it from lysosomal degradation, thus extending its half-life [43]. Amongst these, existing literature provides an in-depth exploration of the biological roles of GP60 and SPARC that utilize albumin as a carrier for antitumor drug delivery [44,46,52].

4.2. Glycoprotein (GP) 60-mediated albumin transport

The gene GP60 (albondin) has been identified to encode a 60 kDa glycoprotein. These glycoproteins are widely distributed throughout the human body but are selectively expressed in microvascular endothelial cells, except in the brain. The key function of GP60 receptors is to specifically bind to the native albumin, followed by the uptake of albumin and its subsequent transcytosis [46,52]. Literature suggests that nearly half of the albumin escapes from the lumen of the capillary via the GP60 receptor, and the remaining crosses the barrier via the extracellular junctions or the fluid phase mechanisms. As shown in Figure 2, internalization of albondin occurs via caveolin-dependent endocytosis followed by subsequent transcytosis without undergoing the degradative endosome-lysosome pathway [52–55]. GP60 functions as a key albumin receptor, demonstrating significant overexpression in tumor tissues and effectively mediating albumin transcytosis in endothelial cells. The above-mentioned functions of GP60 make it an incredible therapeutic target in tumor tissues and offer a valuable basis for designing drug carriers that harness the albumin – GP60 interaction [46]. Nyman et al. reported that ABI-007 was developed to deliver paclitaxel to tumors by leveraging albumin-GP60 interaction [56]. Subsequently, Abraxane^â, an anticancer drug containing paclitaxel, was approved by the U.S. Food and Drug Administration in 2005, which exploited the albumin-GP60 pathway [57,58]. Recently, the interactions between GP60 and albumin have gained traction as it proves to be a promising strategy for advanced imaging techniques, overcoming drug resistance, and enhancing targeted drug delivery [52]. Various studies have provided promising functional evidence for the role of GP60 in albumin transcytosis. Meng et al. utilized a reverse-QTY protein design to make HSA more hydrophobic. This was done by replacing hydrophilic amino acids such as glutamine (Q), threonine (T), and tyrosine (Y) in the helical subdomains of HSA with hydrophobic leucine (L), valine (V), and phenylalanine (F). It showed improved GP60-mediated uptake, tumor targeting, and antitumor efficacy [59]. Another study demonstrated that activation of GP60 increased the transport of albumin by 2.5-fold without affecting fluid permeability. It also depicted that elevated concentrations of albumin hindered the transport of ^{1 2 5} I-albumin, thus confirming GP60 as a specific, saturable receptor critical for albumin internalization [60].

Figure 2.

Albumin-loaded nanoparticles interact with a GP60 receptor on endothelial cells, activating caveolin-1 and facilitating their internalization into the tumor microenvironment. Once there, they encounter SPARC, which enhances their retention and accumulation in the tumor. SPARC also guides the nanoparticles toward tumor cells, where they can be internalized through macropinocytosis.

4.3. SPARC-Driven albumin uptake

Like the GP60 receptor, SPARC is also an albumin-binding protein highly expressed in some cancers [43]. SPARC is also known as basement membrane 40 (BM40) or osteonectin, as it was formerly thought to be expressed in mineralized tissue and was later found to be expressed in both mineralized and non-mineralized tissues [61]. SPARC is a 32 kDa calcium binding-matricellular protein that encodes a single gene that produces a monomeric, secreted glycosylated polypeptide [61]. The open reading frame (ORF) of the SPARC gene encodes a protein with a 17-amino acid hydrophobic signal peptide removed during processing. The mature SPARC protein is composed of three structural domains, each having biological functions specific to its secondary structure [61]. The first domain is an N-terminal calcium binding domain with 52 amino acids (Ala1-Glu52) that possess low affinity and are highly acidic. It inhibits cell spreading, decreases chemotaxis, elevates plasminogen activator inhibitor-1, and suppresses the levels of fibronectin and thrombospondin-1. The second domain is a cysteine-rich follistatin-like domain (Asn53–Pro137) comprising 85 amino acids between the N-terminus and extracellular-calcium binding domains. It contains ten cysteine residues that form disulfide bonds, which help stabilize weakly interacting molecules; the domain interrupts focal adhesions and further enhances angiogenesis and cell proliferation. The third domain is an extracellular calcium-binding domain, consisting of 149 amino acid residues located at the C-terminus (from Cys138 to Ile286), which binds calcium with high affinity. This region induces matrix metalloproteinases, interactions between cells and matrix, and avoids cell spreading, proliferation, and adhesion [62].

SPARC exhibits various biological functions, including its role in interacting with albumin and regulation of cell proliferation and cell differentiation. However, SPARC has depicted four key functions that are identified for the occurrence and development of tumors. The first is its anti-adhesive effect, where SPARC does not allow the cells to bind tightly to the extracellular matrix (ECM). This promotes the tumor cells to separate and invade the surrounding tissues, thus leading to metastasis. Its second function is to degrade the ECM by inducing the synthesis of enzymes like collagenase and gelatinase, which will weaken its barrier function and promote the spread of tumors. Thirdly, SPARC impacts various signaling pathways like PI3K – Akt – mTOR and MAPK pathway, which is majorly involved in cell survival and growth. Lastly, SPARC promotes angiogenesis by aiding in basement membrane lysis and movement of the endothelial cells, further supporting tumor growth and metastasis. Overexpression of SPARC in aggressive tumors suggests its role in disrupting the homeostasis between tumor cells and their surrounding environment [46].

While SPARC is the biomarker potentiating albumin mediated transcytosis, preclinical studies have demonstrated SPARC-independent tumor uptake of nab-paclitaxel in genetically engineered mouse model of pancreatic cancer. The authors found that accumulation and antitumor efficacy of nab-paclitaxel were dose-dependent and were independent of SPARC [63]. Shao et al. highlighted a similar inference that SPARC expression is not precisely required for improved antitumor efficacy of Abraxane in lung cancer models. The study revealed substantial antitumor activity of Abraxane in SPARC-negative tumors, and the effectiveness between Abraxane and Taxol was not associated with SPARC status [64].

4.4. Tumor-targeted delivery of albumin nanoparticles via GP60 and SPARC receptors

As illustrated in (Figure 2), albumin nanoparticles introduced via intravenous (IV) administration use GP60 receptors to cross vascular endothelial cells and access the tumor microenvironment. Once the albumin nanoparticles reach the tumor interstitium, they interact and bind with the SPARC receptors, which are overexpressed by the tumor tissues. This process allows the nanoparticles to deliver their therapeutic agents directly to the tumor site, making treatment a more effective therapeutic strategy [46].

Figure 3 emphasizes the crucial role of KRAS-mutant tumor cells, which have increased macropinocytosis. This means they are likely to take up the albumin nanoparticles more effectively than KRAS wild-type cells. This approach could improve the targeting of cancer cells, particularly in KRAS-mutant cells, which heavily rely on macropinocytosis for nutrient intake. Introducing an albumin nanoparticle into tumor cells undergoing macropinocytosis [34], especially KRAS-mutant cells, the albumin coating on the drug would be recognized as a protein, making it an ideal candidate for macropinocytosis. The tumor cell would engulf the albumin-coated drug and other extracellular fluid and proteins into macropinosomes at the plasma membrane. Once inside the cell, macropinosomes containing the albumin nanoparticles travel through the cytoplasm. These vesicles often fuse with lysosomes, which are responsible for degrading proteins into their constituent amino acids. In the lysosomes, the albumin coating is degraded by proteolytic enzymes. As the albumin is broken down, the drug is likely to be released in its active form into the intracellular environment. Once the drug is released, it could exert its therapeutic effect directly within the cytoplasm or other organelles of the tumor cell, depending on its mechanism of action [65]. A study investigating the uptake mechanism of albumin nanoparticles in KRAS-mutant cells found that FITC-labeled albumin was internalized via macropinocytosis and were co-localized with TMR-dextran. This uptake was observed in cancer cells harboring oncogenic KRAS mutations – NIH 3T3, MIA PaCa-2, and T24 cells, and was significantly reduced by 5-(N-ethyl-N-isopropyl)amiloride (EIPA) treatment, confirming that albumin is taken up via macropinocytosis in these cancer models [31]. Table 1 depicts the overview of drug delivery systems in KRAS wild- and mutant-type cancer cells.

Figure 3.

The illustration shows that KRAS-mutant cells with increased macropinocytosis can take up albumin-loaded nanoparticles effectively. This method could improve cancer cell targeting, especially in KRAS-mutant cells. The albumin coating on the drug makes it an ideal candidate for macropinocytosis, allowing the drug to be released and exert its therapeutic effect directly within the tumor cell.

Table 1.

Overview of drug delivery system for KRAS wild- and mutant-type cells.

Cells	Drug delivery system	Comment	Reference
MIA PaCa-2 BxPC-3	Albumin-drug conjugate Doxorubicin (DOX)	KRAS-driven macropinocytosis and reduced FcRn expression enhance PDAC sensitivity to DOX-ALB, but not free DOX.	[38]
Mouse KPC cells	Albumin bound paclitaxel (nab-paclitaxel, nab-PTX) in combination with gemcitabine	Improved by reprogramming nutrient signaling and enhancing macropinocytosis in cancer cells. Increased macrophage activation and target immune evasion in pancreatic cancer	[50,66]
KRAS mut MIAPaCa-2 cells and wt BxPC-3 cells	Albumin and peptide [HSA to deliver human β-defensin-2 (DF)]	The internalization of DF-HSA in MIAPaCa-2 is apparently mediated by macropinocytosis	[67,68]
MDA-MB-231, MDA-MB-468	Albumin and fluorescent bound nanoparticles: FITC-NP and FITC-BSA	Cross-linked albumin nanoparticles demonstrate a significant increase in uptake within oncogenic KRAS cancer cells when compared to control wild-type KRAS cells. This finding underscores the potential of these nanoparticles in targeted cancer therapies.	[35]
MIA PaCa-2 BxPC-3 AsPC-1 SW1990	Albumin nanoparticle formulation prodrug of β-lap, nab-(pro-β-lap)	Albumin achieves targeted delivery in PDAC due to KRAS-enhanced macropinocytosis and reduced FcRn expression. nab-(pro-β-lap) uptake was profoundly enhanced by KRAS mutation, enhanced safety.	[69]

4.5. FDA-approved albumin nanoparticles

Nanoparticle albumin-bound paclitaxel (nab-PTX), or Abraxane, is a widely used nanomedicine that is prescribed in the treatment of breast cancer, NSCLC, and adenocarcinomas of the pancreas [58]. It was developed to enhance the therapeutic efficacy of traditional taxanes like paclitaxel and docetaxel while minimizing associated toxicities. Its solvent-free formulation avoids the harmful excipients found in conventional taxane preparations, contributing to a more favorable safety profile. Preclinical studies suggest that nab-paclitaxel enhances gemcitabine uptake in pancreatic cancer, warranting further investigation through large clinical trials that collect tumor samples [65]. This formulation reportedly has pharmacokinetic advantages, such as a larger volume of distribution, more rapid clearance, and a higher fraction of unbound drug compared to solvent-based Paclitaxel (sb-paclitaxel). The unique properties of albumin in nab-paclitaxel facilitate better tumor uptake, likely leveraging the enhanced permeability and retention (EPR) effect and receptor-mediated transcytosis across endothelial cells. Additionally, the ability of nab-paclitaxel to cross endothelial barriers is significantly higher than that of sb-paclitaxel, further optimizing its delivery to tumor sites [65].

Li et al. discussed that the delivery and efficacy of the marketed formulation, nab-PTX, was influenced by cancer-cell RAS signaling and macropinocytosis within the tumor microenvironment. This research emphasized the critical role of RAS signaling in nab-PTX uptake, demonstrating that inhibition of the RAS/RAF/MEK/ERK pathway can significantly reduce drug effectiveness. The study reveals that these effects are specific to cross-linked nab-PTX (covalent cross-linking is purposefully introduced in this study to enhance the stability of the nanoparticle and prevent disaggregation) and do not apply to standard paclitaxel (sb-PTX), underscoring the importance of formulation in drug efficacy. The findings suggest broader applications for other albumin-bound therapies and highlight potential combinations with MAPK inhibitors, gemcitabine, or oxaliplatin in clinical trials [50]. Similarly, Wang et al. also successfully reported the potential of folate receptor-directed, macropinocytosis-enhanced human serum albumin-apoprotein of lidamycin-active enediyne for targeting KRAS mutant pancreatic cancer that effectively demonstrated specificity in localization of the tumor, potential cytotoxicity, and high therapeutic efficacy in preclinical models [70].

Fyarro® (nab-sirolimus) is another FDA approved albumin nanoparticle that uses the nab technology. It is an intravenous mTOR inhibitor that was approved in November 2021 for treating unresectable or metastatic malignant perivascular epithelioid cell tumor [71]. This formulation demonstrated improved intratumoral accumulation, effective inhibition of mTOR, and enhanced suppression of tumor growth compared to oral mTOR inhibitors [72].

5. Role of macropinocytosis in lipid nanoparticles for delivery of DNA and RNA

Compared to conventional drugs, nucleic acids such as mRNA, DNA, microRNA (miRNA), or small interfering RNA (siRNA) have garnered attention as one of the emerging therapeutic modalities. These promising genetic modalities have proven advantageous for undruggable genetic disorders, viral infections, and various cancers in terms of high specificity and mechanism of action [73]. Holding an outstanding property different than other modalities, genetic payload with a single dose can give a curative and durable effect. However, nucleic acid has its own in vivo stability and delivery challenges, including its delivery to the specific target site, immunogenicity, and inefficient uptake of the DNA or RNA into the cells [74]. Owing to hydrophilicity, rapid cell clearance, poor permeability into the cell membrane, and high molecular weight of nucleic acids remains an underlying challenge to its potential use. The physicochemical properties of DNA or RNA bring fundamental challenges to its successful delivery mainly due to its negative charge and larger size [73,75]. When targeting RNA for delivery, frequent administration is necessary compared to DNA. This is because the shorter retention time inside the cell results in brief activity. Frequent doses can ultimately lead to toxicity from both the carrier and RNA payload. To modulate and address unmet needs, nanoscale delivery systems have been widely explored for RNA- or DNA-based therapies, broadening biomedical applications. These therapeutic modalities have enabled breakthroughs in vaccine formulations, bioanalysis, protein replacement, and gene silencing [73].

To enhance the delivery of nucleic acids, various technologies, including peptides, lipids, lipid nanoparticles (LNPs), and polymers, have been explored [75]. LNPs are promising candidates for combating various medical conditions by delivering proteins and nucleic acids. They enable precise delivery of gene therapies (DNA or RNA), immunotherapies, or chemotherapeutic agents to targeted tumor cells [76]. LNPs provide an unprecedented opportunity to overcome the hindrance associated with the targeted delivery of nucleic acid by avoiding renal excretion and increasing blood circulation time, securing them from degradation through endosomal escape. Once nucleic acid enters the cell, it goes under enzymatic degradation. In the case of lipid nanoparticles, it will promote the endosomal escape to protect the nucleic acid [76].

Lipid nanoparticles have been utilized to deliver siRNA, leading to the development of Onpattro (Patisiran), the first FDA-approved product using this technology, for the treatment of hereditary transthyretin (TTR) amyloidosis [76]. The most successful LNP formulation for targeting hepatocytes, marketed as Onpattro, consists of four distinct lipids: Dlin-MC3-DMA, an ionizable lipid; DMG-PEG, a PEG-based lipid; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), a helper lipid; and cholesterol [75]. The success of LNPs was further advanced during the COVID-19 pandemic. LNPs were employed as a delivery vehicle in the development of two COVID-19 vaccines of mRNA from Pfizer and Moderna encoding the SARS-CoV-2 spike protein. Worldwide regulatory agencies approved these two vaccines, showcasing their high efficacy [76]. LNPs gained momentum following the success of products like Onpattro, paving the way for developing LNPs for targeting cancer cells. Since the success of Onpattro, which utilized the ionizable lipid Dlin-MC3-DMA, LNPs with ionizable lipids have been extensively explored for targeting various tumoral cells. The charge of the ionizable lipid at physiological pH is neutral and at lower pH found within endosomes and tumor microenvironment is positive. The delivery efficiency and biological performance of LNPs formed with ionizable lipids are heavily influenced by the pKa of the lipid. The pKa value of ionizable lipids determines their ionization and influences their ability to interact with the inner leaflet of the endosomal membrane, which acts as a critical factor in their function. This interaction is essential for destabilizing the membrane and successfully releasing nucleic acids. However, the dynamic pH changes along the endosomal pathway pose challenges in protecting DNA/RNA once they are inside the cell. Via pioneering research, additional pathways for nucleic acids were investigated, facilitating their cellular entry and trafficking in the cell. Clathrin-mediated endocytosis, phagocytosis, macropinocytosis, and caveolae-mediated endocytosis pathways were scrutinized to protect the DNA/RNA from degradation and improve their efficacy and stability inside the cell within the cytoplasm. Specifically, macropinocytosis is known for its bulk uptake of extracellular fluid driven by actin-mediated membrane ruffles. Macropinocytosis regulators located at the plasma membrane play a key role in trafficking DNA/RNA encapsulated in LNPs to specific locations within the cell once internalized into macropinosomes. After entry, these macropinosomes typically follow the classical late endosomal/lysosomal pathway. As a result, the nucleic acids may either undergo metabolic processing or be marked for degradation, depending on the intracellular signaling and functional requirements [75].

With the vast majority of oncogenic proteins remaining undruggable by small molecules, the use of siRNA for knocking down key proteins has been very successful in various preclinical studies. RNA interference (RNAi) using siRNAs to target mutant KRAS alleles offers a promising therapeutic strategy, particularly for KRAS-mutant lung cancers. However, a significant barrier to clinical application is the lack of efficient and biocompatible siRNA delivery systems. To overcome this, Mehta et al. developed bovine serum albumin (BSA) nanoparticles to deliver siRNA specifically targeting the KRAS G12S mutation. These nanoparticles enable endosomal escape, potent KRAS knockdown, and reduced cancer cell growth. This knockdown led to a reduction in cell growth in KRAS G12S mutant lung adenocarcinoma cells. Overall, BSA nanoparticles loaded with siRNA specific to mutant KRAS offer a promising therapeutic strategy for treating cancers with KRAS mutations. This approach effectively addresses challenges associated with RNAi-based treatments and is currently being evaluated in vivo in research studies [77]. As discussed earlier, siRNA can selectively silence specific oncogenic drivers in different cancer cells by recognizing and binding to specific messenger RNAs (mRNA). This binding results in the formation of a catalytic complex with an RNA-induced silencing complex (RISC), and this process effectively degrades the targeted mRNA by preventing protein synthesis [78]. siRNA to silence driver mutations remains largely untapped due to its challenging delivery, which is attributed to its fragility as a non-viral vector. To overcome this, protein-based nanocarriers, particularly albumin-based nanocarriers, are widely explored due to their promising potential in targeted nucleic acid delivery and evading immune system responses. Due to its highly negatively charged surface, albumin is highly water-soluble [79,80]. GP60 and SPARC are albumin-binding proteins, while Cav-1, a protein responsible for albumin endocytosis, is upregulated in cancer cells, promoting albumin accumulation within tumors [78]. As a result, albumin penetrates rapidly dividing tumor cells at a rate > 4 times higher than nanocarriers, meeting their high metabolic demand [80]. Albumin-based nanoparticles are commonly employed to deliver nucleic acid, including siRNAs, plasmids, and oligonucleotides. To improve the internalization of siRNA using HSA as a carrier, Son and coworkers experimented by introducing a thiol moiety into vascular endothelial growth factor (VEGF) siRNAs and HSA. This modification leads to the formation of stable nanoparticles through self-crosslinking, with the thiol groups helping to improve the binding affinity between siRNA and albumin, resulting in a more robust structure. As a result, albumin-based nanoparticles accumulated in tumor areas in prostate cancer-3 (PC-3) xenograft models, demonstrating significant efficacy by reducing tumor volume by 80% compared to free thiol siRNAs, which were rapidly excreted through the kidneys. In another study conducted by Xu and coworkers to suppress tumor cell proliferation in liver carcinoma and to enhance their recognition by human hepatocellular carcinoma, researchers coated siRNA-chitosan complexes with detachable, pH-responsive BSA. This BSA coating on chitosan nanoparticles significantly improved mRNA silencing efficiency, increasing it from 46.9% to 61.8%. Cheng and coworkers developed HSA nanoparticles functionalized with stearyl polyethylenimine (PEI), which binds noncovalently with siRNA and plasmid (CRISPR/Cas9). This complex exhibited a synergistic effect by silencing the expression of programmed cell death ligand-1 (PD-L1) and inhibiting it by 21.2% for cancer immunotherapy [78]. A plasmid encoding the melanoma differentiation-associated gene hMDA-7 was utilized for pancreatic cancer therapy. To target BXPC-3 cells, hMDA-7 was encapsulated in BSA nanoparticles, resulting in a 25.6% apoptosis rate compared to 15.3% with direct plasmid administration. This formulation was further assessed in a mouse xenograft model where it led to the downregulation of Matrix Metalloproteinase-9 (MMP-9), VEGF, and Matrix Metalloproteinase-2 (MMP-2) proteins, ultimately leading to tumor growth suppression [81]. Wartlick and coworkers developed antisense oligonucleotide (ASO) loaded nanoparticles using HSA, significantly enhancing cellular internalization without inducing notable toxicity in MDA-MB-435 and MCF-7 cells [78]. Another experiment utilizing HSA as a coating material in KB human oral carcinoma cells was conducted. Nanoparticles were loaded with antisense oligonucleotide targeting Bcl-2 protein. The efficiency of the Bcl-2 downregulation depended on the molar ratio of HSA in the formulation, suggesting that an increment in HSA concentration decreased the silencing efficiency of Bcl-2. The HSA: liposome 3:1000 molar ratio gave optimum down-regulation of Bcl-2 [82]. Similarly, in another study, breast cancer cells and a corresponding xenograft mouse model were treated with siRNA-loaded lipid nanoparticles coated with HSA (HSA-LNPs-siRNA) to target green fluorescent protein (GFP). Compared to uncoated nanoparticles, HSA-coated nanoparticles significantly reduced the GFP fluorescence in breast cancer cells. Similarly, after systemic administration of HSA-coated nanoparticles in the animal model, a 37% reduction in GFP expression was observed [83]. Specifically for triple-negative breast cancer (TNBC) cells, Sarett and coworkers used HSA as a carrier in vivo for modified siRNAs with a diacyl lipid moiety (siRNA-L2), resulting in increased pharmacokinetic properties of siRNA. Compared to commercial nanocarrier jet PEI, this HSA-based nanoconjugate exhibited a 46-fold increase in cellular uptake and 19-fold higher accumulation in tumor cells within a mouse orthotopic model of human TNBC [84]. In another study on TNBC, albumin-binding siRNA exhibited higher efficacy for TNBC therapy compared to a small molecule inhibitor of Myeloid cell leukemia-1 (MCL-1) designed for in situ albumin binding [80]. Miao et al. formulated a LNPs with a modified ionizable lipid – Dlin-MC3-DMA in combination with alkyne lipids containing amine groups. This formulation enhanced mRNA delivery and identified an ApoE independent cellular uptake pathway for LNPs involving albumin associated macropinocytosis and endocytosis in the liver [85]. On the similar lines, Tan et al. developed cabazitaxel loaded human serum albumin nanoparticles (CTX-HSA-NPs) which exerted enhanced anti-tumor activity and reduced toxicity compared to cabazitaxel-tween. In addition, they formulated TGFβ-1 siRNA loaded lipid nanoparticles and combined them with CTX-HSA-NPs yielding improved antitumor efficacy both in vivo and in vitro in a paclitaxel resistant non-small cell lung cancer model [86]. Albumin-based nanocarriers offer several advantages, including possible scaleup options, reproducibility, and easy production without causing undesired interaction with serum components. Hence, albumin is widely utilized for delivering immunomodulatory agents, biologics, and chemotherapeutics as anti-cancer therapies in pharmaceutical applications.

6. Crosstalk between KRAS pathways and drug delivery systems

Despite years of research, targeting KRAS has been difficult due to the intrinsic characteristics of these proteins. This undruggable target has proven to be a central driver in different cancer types, with only sotorasib and adagrasib being approved by the FDA against KRAS G12C mutant [32]. A significant impact has been observed in the delivery of drugs due to the changes in the cell surface receptor expression, altered cellular uptake mechanism, and evolving tumor microenvironment [87]. Recent studies have identified macropinocytosis, a nutrient-scavenging endocytic process, a critical mechanism KRAS-mutant cancer cells utilize to thrive under nutrient-depleted conditions. Hanada et al. show that KRAS-mutant colorectal tumoral cells majorly rely on macropinocytosis, driven by Rac1 and PI3K, to survive in nutrient-poor conditions. This process was found to be closely related to asparagine metabolism. Blocking macropinocytosis and asparagine availability has significantly suppressed tumor growth in vivo, proposing a new therapeutic avenue for KRAS-mutant cancers [88]. Another strategy involving the convergence of the KRAS pathway and an effective drug delivery system is mitigating drug resistance. Various genomic and histological factors can hinder the efficacy of covalent KRASG12C inhibitors. This highlights the necessity for innovative treatment approaches to delay and overcome resistance to this medication in cancer patients [89], in addition to exploring formulations and combinatorial therapies [90].

Hyperactivation of KRAS can primarily lead to the enhanced proliferation of the tumoral cells, irrespective of the treatment, by engaging in PI3K/Akt and Raf/MEK/ERK pathways, where the signaling is compensated [91]. The combination therapy is where multiple inhibitors are co-delivered via nanoparticulate systems that can block different nodes in the KRAS network at the same time, thus preventing the inception of survival pathways for the KRAS mutant cancers [92]. Amodio et al. reported that targeting EGFR and KRAS^G12C is a highly effective combination therapy in patients with CRC and patient-derived organoids [93]. Significant interest has been observed in research that uses a combinatorial approach of targeted therapies with immunotherapy in solid tumors. Preclinical data have ignited curiosity in understanding synergistic action when the KRAS^G12C inhibitor is used along with immune checkpoint inhibitors like atezolizumab and pembrolizumab [92,94,95]. Predictive biomarkers can provide patients with personalized medicine that specifically targets KRAS mutations using combination therapies [96]. Gu et al. demonstrated a novel approach that leveraged the synergistic effect of gene silencing and chemotherapy in KRAS-mutant, p53-deficient NSCLC. siKRAS, miR-34a, and cisplatin were co-delivered by developing layer-by-layer nanoparticles. This study showed a promising RNA-chemotherapeutic combinatorial strategy depicting enhanced in vitro cytotoxicity, preferential cell uptake in the lungs of the tumor-bearing mice, and an increase in the survival rate [97]. Figure 4 summarizes the critical interplay of advanced drug delivery systems and KRAS pathways involved in cancer progression.

Figure 4.

Crosstalk of drug delivery systems in the KRAS pathway plays a pivotal role in cancer progression. Nanoparticle-based approaches can offer innovative ways to enhance the efficacy, specificity, and safety of KRAS-targeted therapies.

7. Challenges and future perspectives

Various challenges have been associated with leveraging KRAS mutation-mediated macropinocytosis for cancer therapy. One significant challenge in leveraging macropinocytosis for cancer nanomedicine is the observed heterogeneity in macropinocytosis levels across various KRAS mutations. Different KRAS mutations can lead to distinct cellular behaviors, metabolic adaptations, and signaling pathways, affecting how effectively macropinocytosis is activated. KRAS mutations G12D and G12V enhance the process of macropinocytosis. Meanwhile, G12R shows reduced macropinocytic activity due to structural changes that affect its interaction with proteins like PI3K12 [98]. This variability complicates the development of universal therapeutic strategies, as a one-size-fits-all approach (a single nanocarrier strategy) may not be effective. Understanding the mechanistic underpinnings of these differences is crucial for tailoring therapies that exploit macropinocytosis in a mutation-specific manner [18,98]. Researchers reported that combining MEK inhibitors with gemcitabine/Nab-paclitaxel reduced tumor growth in PDX models but could induce secondary KRAS mutations, driving tumor heterogeneity. This heterogeneity created therapy-resistant subclones, hampering the effectiveness of the treatment against KRAS-mutant cancers [91].

Additionally, targeting macropinocytosis in cancer therapy poses a challenge. It also shows activity in normal cells, preventing selective inhibition toward cancer cells without harming healthy cells [34]. Although albumin nanoparticles offer a promising approach for targeting KRAS-mutant cancers; its clinical translation is challenged by several potential side effects. One of the primary concerns is competing with endogenous albumin, which is abundant in human plasma at concentrations (nearly 35–50 g/L). The endogenous albumin can compete with albumin nanoparticles for receptor-mediated uptake via gp60 and SPARC, causing saturation of the common pathways and reduction in the internalization of nanoparticles in KRAS-mutant cells [99,100]. Additionally, gp60 and SPARC receptors are also expressed in healthy tissues, leading to nonspecific uptake of albumin nanoparticles that may reduce tumor selectivity and increase systemic side effects [35]. Moreover, while albumin is usually non-immunogenic, elevated doses or repeated administration of albumin nanoparticles have been proven to showcase mild inflammatory responses, including higher cytokine levels and immune cell infiltration in preclinical models [101]. The use of serum albumin from non-human sources – such as bovine serum albumin (BSA) – can provoke immune responses in both humans and mice, and proteins like ovalbumin are well-known for their ability to stimulate the immune system. This emphasizes on HSA that proves to be the most clinically relevant albumin for therapeutic drug delivery applications [44]. The clinical relevance of EPR-based nanomedicine faces challenges, including the tumor stroma, impaired lymphatic drainage, and differences in nanoparticle uptake among tumor sites. Despite these limitations, understanding the biology of solid tumors is crucial for designing effective, targeted drug delivery systems in oncology. The EPR effect and its clinical relevance are inconsistent due to heterogeneity in human tumors and patient-specific factors [49]. Future research should focus on characterizing the macropinocytic profiles of various KRAS mutations to facilitate the design of more personalized and effective nanomedicine approaches.

8. Conclusion

KRAS oncogenic mutation is widespread in various solid tumors. Macropinocytosis, nutrient-scavenging endocytic process, is one of the signature characteristics of KRAS mutant tumors. KRAS-Albumin crosstalk could potentially be explored by utilizing albumin-coated nanomedicine as a trojan horse for the delivery of anticancer molecules and nucleic acid. By leveraging the unique properties of albumin and its ability to enhance drug delivery through macropinocytosis, researchers are developing targeted therapies that may improve treatment outcomes for patients with these challenging malignancies. However, significant hurdles remain, particularly the heterogeneity in macropinocytosis levels associated with different KRAS mutations, which complicates the design of universal therapeutic approaches. Additionally, the potential for combining macropinocytosis-targeted therapies with KRAS inhibitors introduces complexities related to drug resistance and optimal delivery strategies. Ultimately, advancing this research holds the potential to significantly enhance patient outcomes and transform the landscape of cancer therapeutics.

Funding Statement

This work was supported by the National Institutes of Health: NIH/NIGMS/R16 [1R16GM145556-01A1].

Article highlights

• KRAS mutation enhance macropinocytosis in cancer cells for nutrition uptake.

• Albumin coated nanoparticle could be useful for delivering anticancer medicine

Author contributions

Drishti Rathod: Conceptualization, Methodology, Investigation, Writing – original draft. Poonam Gawali: Visualization, Writing – review & editing. Bhoomi Dholariya: Writing – original draft. Mukti Vats: Writing – original draft. Osaretin Otagho: Writing – original draft. Ketan Patel: Conceptualization, Investigation, Supervision, Writing – review & editing.

Disclosure statement

Ketan Patel is a member of the Nanomedicine Editorial Board. They were not involved in any editorial decisions related to the publication of this article, and author details were not made available to the article’s peer reviewers as per the journal’s double-anonymized peer review policy.

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Reviewer disclosure

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.