Tumor-targeted miRNA nanomedicine for overcoming challenges in immunity and therapeutic resistance

Abstract

miRNA are critical messengers in the tumor microenvironment (TME) that influence various processes leading to immune suppression, tumor progression, metastasis and resistance. Strategies to modulate miRNAs in the TME have important implications in overcoming these challenges. However, miR delivery to specific cells in the TME has been challenging. This review discusses nanomedicine strategies to achieve cell-specific delivery of miRNAs. The key goal of delivery is to activate the tumor immune landscape as well as to prevent chemotherapy resistance. Specifically, the use of hyaluronic acid-based nanoparticle miRNA delivery to the TME is discussed. The discussion is focused on miRNA-125b for reprogramming tumor-associated macrophages to overcome immunosuppression and miRNA-let-7b to overcome resistance to anticancer chemotherapeutics because both these miRNAs have been extensively evaluated for delivery with hyaluronic acid-based delivery systems.

Plain language summary

miRNAs are the messenger molecules with the tumor that have significant influence on the cancer growth and progression. Many strategies have been evaluated to modulate these messengers artificially to obstruct cancer growth and destroy cancer cells. This review discusses one such strategy to deliver these messenger miRNAs using hyaluronic acid-based nanoparticles that harness the body's own immune system to fight cancer. The two miRNAs that this review discusses are miRNA-125b and miRNA-let7b.

Graphical abstract

Cancer development involves a series of events in which normal cells first convert to rapidly growing cells through a succession of genetic events [1]. However, these rapidly growing cells need a favorable environment to progress to cancer. This favorable environment for tumor cell progression, growth and metastasis is termed the tumor microenvironment (TME) [2]. The rapid progress of scientific knowledge regarding the mechanisms of cancer progression has led to updating the hallmarks of cancers in the scientific community every decade. With the evidence of the existence of different cell types within the tumors, the TME found its place within the hallmarks of cancer in the past decade [3]. These hallmarks of cancers clearly illustrate the importance of the microenvironment in tumor growth and metastasis and in determining a potential therapeutic's efficacy in inhibiting tumor growth [3]. The TME is a dense environment comprising varied cell types such as tumor cells, cancer stem cells, stromal cells (fibroblasts, endothelial cells, adipocytes) and immune cells such as macrophages, dendritic cells and T cells; and the complex interactions between components [4]. The noncellular components of the microenvironment within the tumor include extracellular matrix such as collagen, fibronectin, hyaluronan and laminin. The communication between the tumor cells and the nonmalignant components of the tumor environment is an important factor in tumor development and progression [5].

TME defines the immune landscape & resistance to cancer therapeutics

The diverse TME is one of the leading causes of heterogenicity observed in tumors of similar origin [6]. Heterogenicity among tumors often leads to variance in treatment efficacy among patients [7]. The most widespread example of TME heterogenicity is the occurrence of cold and hot tumors, which solely depends on immune cell populations in the TME [8]. TMEs with an increased population of infiltrating T cells are termed hot tumors, whereas TMEs with a low numbers of infiltrating T cells and increased population of Tregs and/or immune-suppressive cells are termed cold tumors. Thus, the TME plays a major role in immunomodulation [9] and the development of resistance to cancer therapeutics [10,11].

The nonmalignant cellular elements of the TME are significant contributors to antitumor immunity and therapeutics resistance. The nonmalignant cellular components include immune cells, stromal cells, ECM and other secretory molecules. Among immune cells, macrophages are one of the important cell types that undergo a phenotype switch in the TME (termed M2/anti-inflammatory macrophages), in turn supporting tumor development and progression and cancer metastasis [12]. The tumor-associated macrophages (TAMs) are known to induce resistance to cancer therapeutics through multiple mechanisms. TAMs secrete TGF and TNF-α to activate the epithelial to mesenchymal transition (EMT) pathway, in turn inducing EMT in tumor cells and resistance to therapeutics [13]. Additionally, TAMs secrete angiogenesis factors, such as VEGF, thymidine phosphorylates, urokinase-type plasminogen activator and phosphatidylinositol-glycan biosynthesis class F protein in tumor-promoting tumor progression and metastasis [14]. Furthermore, TAMs have been identified for secreting immunosuppressive factors such as prostaglandin E2, IL-10, CCL18, CCL22 and TGF-β, which play a major role in inhibiting Th1 immune responses [15]. Similar to macrophages, neutrophils are another class of immune cells of the TME that show changes in phenotype to perform immunosuppressive functions by recruiting macrophages and Tregs [16]. Additionally, the myeloid-derived suppressors cells are immune cells of the TME that are also involved in inducing immune suppression and resistance to cancer therapeutics [17].

The next class of nonmalignant cellular components of the TME is the stromal cells. Fibroblasts that are stromal cells in the TME mainly remodel the extracellular matrix to promote tumor development and progression [18]. Fibroblasts are involved in the activation of the Wnt signaling pathway, inducing resistance to cancer therapeutics through activation of p-glycoproteins and reducing intracellular reactive oxygen species [19]. Mesenchymal stem cells (MSCs) are yet another stromal cell type involved in promoting tumor progression by inducing resistance to cancer therapeutics [20–22]. These stem cells are also involved in recruiting anti-inflammatory macrophages in the TME [23], increasing the cancer stem cell population [24] and inducing EMT [25]. However, studies have also demonstrated the positive role of MSCs in the TME in multiple cancers and MSC therapy had been evaluated as an anticancer therapy [26,27]. Thus, further detailed evaluation is essential for the role of MSCs in the TME.

The nonmalignant components of the TME include secretory molecules along with the cellular components. Each of the cell types of the TME communicates with the others through direct interactions or paracrine interactions. The secretory molecules for paracrine interactions include growth factors, interferons and interleukins, among others. Another major class of cell-to-cell communications identified over the past decade are exosomes, which contain messenger molecules such as proteins or nucleic acids such as miRNA and mRNA [28]. This review focuses on modulating miRNAs in the TME to improve antitumor immunity and overcome therapeutic resistance.

Biogenesis & functions of miRNAs

miRNAs are a type of small, noncoding RNAs that have evolved in eukaryotes and regulate multiple biological pathways, such as immune responses, cell cycle progression, differentiation, proliferation, metabolism, apoptosis and stress tolerance [29]. About 2 decades ago, the first miRNA lin-4, which was discovered in Caenorhabditis elegans, was found to have complementary antisense to multiple sites in the 3′ UTR of the gene called lin-14 [30,31]. Since then, there has been much progress in the understanding of the biogenesis, functions and diagnostic potential of miRNA in a variety of diseases. Within the genome, miRNAs are encoded as individual genes or as clusters containing multiple different miRNAs [32]. The biogenesis of miRNA starts with the transcription of miRNA genes by RNA polymerase II/III (Pol II) into pri-miRNAs and is regulated by RNA Pol II-associated transcription factors and epigenetic modulators [33]. Pri-miRNAs are processed to single hairpins termed precursor miRNAs (pre-miRNAs) by a nuclear protein complex called a microprocessor. The microprocessor in a protein complex that contains Drosha (RNase III enzyme), the dsRNA-binding protein DiGeorge critical region 8, and additional components such as the DEAD-box RNA helicases p68 and p72 [34]. These processed pre-miRNAs are then transported to the cytoplasm through the export receptor exportin 5 and are processed by Dicer to 20–25 nucleotides dsRNA [35]. Once in the cytoplasm, the miRNAs exhibit their functional effects on various pathways, which is discussed in the next sections.

miRNAs have been shown to mediate gene regulation through either gene silencing or upregulation of gene expression or translation activation or inducing nuclear post-transcriptional gene regulation. In the case of miRNA-mediated gene silencing, the interaction between miRNA and its target mRNA is facilitated by RISC loading in which the ds miRNA produced by Dicer is transferred to the Argonuate (AGO) protein family. RISC chooses one strand to turn into the mature miRNA (guide strand) and discards the passenger strand [36]. miRNA in the RISC complex drives it to an mRNA target in a complementary-nucleotide-based way, initiating pathways such as mRNA deadenylation, decapping or translation repression, finally inhibiting protein synthesis [37,38]. Generally, the 3′UTR contains the mRNA target site, and the seed sequence is positioned at 2–7 nucleotides on the complementary strand, called the seed sequence [39]. A detailed mechanism of the various regulatory elements involved in the RISC complex and the miR-mediated silencing has been previously reported [34]. Finally, it has been reported that shuttling of miR and AGO by TNRC6A, Argonaute-navigator and their interaction with target mRNA within the nucleus leads to nuclear mRNA degradation, but a detailed mechanism has yet to be established [40,41]. Overall, miRNAs regulate gene expression not only in the cytoplasmic compartment but also in the nuclear compartment through their interaction with the aforementioned RNA–protein complexes.

miRNAs play a pivotal role in the regulation of the TME

Given the role of miRNA in the regulation of gene expression and biological functions of the cell, miRNAs have been shown to have an impact on conventional hallmarks of cancer. Abnormal miRNA expression in cancer cells compared with normal cells has been linked to a variety of mechanisms, such as chromosomal abnormalities, transcriptional control changes, epigenetic changes and defects in the miRNA biogenesis machinery [42,43]. Several studies using miRNA profiling and sequencing of tumor samples have shown that miRNAs can act as either tumor suppressors or oncogenic miRNA depending on whether they are downregulated or upregulated in a variety of cancers.

miRNAs as tumor suppressors

miRNA downregulation in human malignancies causes elevation or aberrant levels of target mRNA expression leading to increased cell growth, apoptosis, metabolism and invasion. In mammalian cells, miRNA-let-7 is the keeper of differentiation, and its unusual regulation and reduced expression of let-7 have been linked to cancer initiation and progression [44]. Let-7 targets oncogenes such as c-Myc, RAS, HMGA2, VEGF, Lin28 MDR family and long noncoding RNA H19 [45]. Recent studies have further shown that let-7 suppresses the expression of PD-L1 through interaction with the 3′-UTR in many human cancer cells, demonstrating that the LIN28/let-7 axis takes part in immune surveillance, driven by tumor cells [46]. miRNA levels can be regulated by tumor suppressor genes, and one such example is miRNA-34a, which is dysregulated in various cancers and has been demonstrated to be directly regulated by tumor suppressor p53 [47]. miRNA-34a overexpression can limit cancer cell growth and tumor progression in non-small-cell lung cancer models [48,49]. miRNA-34a also antagonizes many oncogenic processes by regulating genes that function in various cellular pathways [50]. Similarly, the miRNA-200 family (miRNA-200a, miRNA-200b, miRNA-200c, miRNA-141 and miRNA-429) is one of the widely studied miRNAs and is involved in many diverse functions, such as induction of EMT via downregulation of E-cadherin and consequent increases in ZEB proteins [51]. It has also been demonstrated that miRNA-200 influences angiogenesis indirectly via the downregulation of CXCL1 and IL-8, which are major cytokines in the TME [52].

miRNAs as oncogenes

miRNAs act as oncogenes by targeting tumor suppressor genes and have been reported to be overexpressed in tumor cells compared with normal cells. Two of the major oncogenic miRNAs reported are the miRNA-17-92 cluster and miRNA-21 [53]. Noncoding RNA C13orf25 encodes the miRNA-17-92 cluster and is known to be upregulated in several cancers. Expression levels of several miRNAs are elevated due to the amplification of the 13q31-32 locus in the chromosome in a variety of cancers [54]. Phosphate and tensin homolog (PTEN) and the transcription factor E2 family are among the earliest validated targets of the miRNA-17-92 cluster [55]. More recently miRNA-17-92 cluster has also been shown to regulate a variety of immune cells in the TME, such as B cells and subsets of T cells, such as Th1, Th2, T follicular helper cells, Tregs, monocytes/macrophages, NK cells and dendritic cells [56]. miRNA-21 is one of the trivial biomarkers in many diseases, its overexpression is strongly associated with a variety of hematological and solid malignancies [57]. miRNA-21 exerts its oncogenic function by binding to the 3′UTR of the multiple tumor suppressor genes such as PTEN, TPM1 and PDCD4 [58–60]. It induces inhibition of cellular apoptosis by negatively regulating p53 and NF-κB signaling [61]. miRNA-21 has also been reported to be one of the most abundant miRNAs in TAMs and was shown to drive tumor growth by modulation of cytokine production [62]. Multiple studies have reported that miR-155 promotes growth in several types of cancer, including breast and lung cancers [63]. miRNA-155 directly targets and inhibits many genes, including SHIP1, WEE1, VHL, TP53INP1, BCL2 and SOX family genes, which are involved in many critical cellular functions including immune response, DNA damage response, hypoxia, inflammation and tumorigenesis [64].

Overall, these miRNAs, in addition to cancer cells, also regulate various stromal cells and exert their impact to recruit or promote the differentiation of suppressive immune cells, such as Treg, cancer-associated fibroblasts, tumor-associated MSCs, TAMs and others involved in the TME [65]. These are some of the most investigated miRNAs, but there are certainly many more examples of specific miRNAs in each type of cancer that have been reviewed extensively by others [66,67].

miRs in macrophage phenotype modulation

The percentage of TAMs in the TME is associated with a poor prognosis [68–70]. Given the role of miR in TME, several studies performed over the last decade suggest a prominent role for miRNAs as key regulators of macrophage differentiation, infiltration and activation. Certain miRNAs such as miRNA-155, miRNA-125b, let-7b and miRNA-29, are known to promote repolarization of macrophages from M2 toward M1 phenotype and play a role in the regulation of immune responses in cancer and inflammation [71–73]. It has been reported that the expression of miRNA-21 in TAMs in particular, were responsible for promoting tumor growth [62]. Another study in prostate cancer showed the let-7b upregulation is characteristic of prostatic TAMs and the downregulation of let-7b in TAMs leads to changes in expression profiles of inflammatory cytokines such as IL-12, IL-23, IL-10 and TNF-α [74]. TAMs treated with let-7b inhibitors reduce angiogenesis and prostate carcinoma cell mobility. Although these studies contradict the previously described role of let-7b as a tumor suppressor miRNA in lung and ovarian cancer, it is important to realize that the impact of miRNAs on tumorigenesis has specific functions depending on the type of cancer, cell type and the baseline levels compared with healthy tissues. Table 1 lists miRNAs involved in immunomodulation for various types of cancers; however, the focus of this review is on miRNA-125b and its role in overcoming immunosuppression.

Table 1. . miRNAs involved in immunomodulation in for various types of cancers.

Tumor type	miRNA	Mode of overcoming immune suppression	Refs.
Melanoma	miRNA-23a miRNA-155	Increases CTL-mediated cytotoxicity Induces effector T-cell responses	[75] [76]
Lung	miRNA-34a miRNA-200a,b,c miRNA-760 miRNA-125b	Inhibits PD-L1 expression Inhibits PD-L1 expression Downregulating IDO1 and eliminating Tregs Repolarization of TAMs	[77] [78] [79] [80]
Lymphocytic leukemia	miRNA-93 miRNA-106b	Decreases CXCL12 AND PD-L1 expression Decreases CXCL12 AND PD-L1 expression	[81] [81]
Hepatocellular carcinoma	miRNA-182	Activates NK cells	[82]
Ovarian cancer	miRNA-424 miRNA-125b	Activation of monocytic differentiation, antitumor immune response Repolarization of TAMs	[83] [84]
Colorectal carcinoma	miRNA-138-5p miRNA-let7a	Inhibits PD-L1 expression Decreases immunocompetence by T cells	[85] [86]
Acute myeloid leukemia	miRNA-17-5p, miRNA-20a, miRNA-106a	Inhibition of monocytic differentiation and maturation	[87]
Breast cancer	miRNA-101-3p	Repolarization of TAMs	[88]
B-cell lymphoma	miRNA-152-3p	Promotes cytotoxic T-lymphocyte proliferation via the PD-1/PD-L1 checkpoint	[89]

TAMs: Tumor-associated macrophages.

Changes in miRNA profiles with tumor relapse & chemoresistance

Each tumor type has a distinct miRNA signature that distinguishes it from normal tissues and other cancer types. Advances in various analytical tools such as miRNA profiling using microarray-based hybridization-based methods (e.g., DNA microarrays, quantitative reverse transcription PCR, Nanostring) and high-throughput sequencing (e.g., RNA sequencing and next-generation sequencing) have enabled the profiling of tumor samples from various cancer types. One of the first studies performed in epithelial ovarian cancer patient samples using genome-wide microarray analysis showed that the most significantly overexpressed miRNAs were miRNA-200a, miRNA-141, miRNA-200c and miRNA-200b, whereas miRNA-199a, miRNA-140, miRNA-145 and miRNA-125b were among the most down-modulated miRNAs. miRNA expression levels within the same type of cancer have been found to have differential expression profiles depending on various factors, such as cancer subgroup and stage, response to chemotherapy, disease prognosis and progression-free survival of disease, among others. miRNA profiling revealed differences in miRNA expression between molecular subtypes of cancer such as high-grade serous ovarian carcinoma, clear cell ovarian carcinoma and ovarian surface epithelium. miRNA-200 family members and miR-182-5p were the most overexpressed in high-grade serous ovarian carcinoma and clear cell ovarian carcinoma compared with ovarian surface epithelium, whereas miRNA-383 was the most underexpressed [90]. The same study in ovarian cancer also identified miRNAs expression that was correlated with specific ovarian cancer bio-pathologic features, such as histotype, lymphovascular and organ invasion and involvement of ovarian surface. Similarly, studies performed in surgically resected EGFR-mutated lung adenocarcinoma samples found that miRNA-21 had a significantly higher expression in lung cancer compared with normal lung tissues [91]. A study designed to identify miRNA-based circulating biomarkers to predict clinical outcomes of colorectal cancer shows that a four miRNA signature (miRNA-652-3p, miRNA-342-3p, miRNA-501-3p and miRNA-328-3p) could help in the prediction of tumor relapse and the overall survival of patients [92].

Changes in miRNA expression are associated with the development of resistance to chemotherapy in patients [93,94]. More evidence linking the miRNA and resistance was established when the NCI-60 panel of various cancer cell types was profiled for miRNA expression in the response to a panel of anticancer drugs. The miRNA expression profile revealed a strong correlation between the expression patterns of miRNAs and the potency patterns of anticancer compounds [95]. In particular, the change in cellular levels of the let-7 family, miRNA-16 and miRNA-21 affected the potencies of several anticancer agents by up to 4-fold depending on the compound class and cancer type. miRNAs have been demonstrated to alter cellular response to anticancer agents via modulation of survival pathways and/or apoptotic response or to affect mechanisms other than survival and apoptotic signaling, such as drug targets and DNA repair systems [93]. miRNAs may also function in drug resistance, including miRNA-130a, which targets the prometastatic and chemoresistance associated M-CSF and miRNA-27a and miRNA-451, which regulates the expression of the drug transporter MDR1 (P-glycoprotein), a protein implicated in paclitaxel-resistant ovarian cancer [96]. Exosome-mediated miRNA transfer between host–tumor cells play a pivotal role in tumor progression and resistance to treatment [97]. These studies indicate the role of miRNA profiling in the identification of novel biomarkers, prognostic indicators and therapy for precision medicine in cancer. Table 2 lists miRNAs that have been involved in drug resistance for various types of cancers; however, the focus of this review is on miRNA-let-7b and its role in overcoming drug resistance.

Table 2. . miRNAs involved in drug resistance for various types of cancers.

Tumor type	miRNA	Targets/pathway	Refs.
Ovarian cancer	miRNA-let-7 (b, c, d, e, f, i, g) miRNA-27a miRNA-451 miRNA-200 (a, b, c)	MDR1, Myc, RAS, E2F1, E2F5, LIN28, PBX3, HMGA2, BRCA1, RAD51, PARP, IGF1 HIPK2, AMPK, MDR1, AKT, K-RAS/B-RAF c-Myc, MDR1/P-glycoprotein, RAB14 TUBB3, ZEB1, ZEB2, E-Cadherin, DNMT3A/DNMT3B, AP-2α	[98–102] [103–105] [106–108] [109–112]
Lung cancer	miRNA-34 (a, b, c) miRNA17-92 Cluster (miRNA 17, 20a, 20b) miRNA-21 miRNA-221/222	Wnt1, Notch1, Wnt3, MTA2, CD44, c-MYC, among others (PEBP4, TCF1/LEF1) p53, TGFβR, PTEN, CDKN1A, BCL-2, BECN1 TRAIL, SIDT1, PI3K/AKT APAF-1	[113–116] [117–119] [120,121] [122]
Breast cancer	miRNA-30 b, c miRNA-141 miRNA-17/20 miRNA-155	TWF1, YWHAZ, MAPK pathway, CCNE2 CTNNB1 P53 and AKT, cyclin D1 RAD51, FOXO3a	[123,124] [125] [126,127] [128,129]
Pancreatic cancer	miRNA-320 a, c miRNA-1246 miRNA-210 miRNA-455	PDCD4, SMARCC1 CCNG2 ABCC5 TAZ	[130,131] [132] [133] [134]

Challenges associated with miRNA delivery & transfection

The major challenge with miRNAs as therapeutic modalities has been their effective functional delivery to the cell type of interest. Nanocarriers have been at the forefront as delivery systems for miRNAs [90]. The use of positively charged polymers and lipids have been extensively exploited to interact electrostatically with negatively charged miRNAs, forming a complex capable of protecting miRNAs from degradation activity of the nucleases and delivering them intact to the cells [135]. However, for the miRNAs to demonstrate their desired activity, the nanocarriers need to deliver the miRNAs effectively [136]. The nanocarriers must overcome extracellular barriers such as passive delivery to the liver, and spleen because the permeability of vasculature results in leakage of nanocarriers primarily to these organs. Other extracellular barriers include the endo- and exonucleases in the circulation and stimulation of the immune system for the breakdown of nanocarriers before delivering the nucleic acid to the site of action [137].

As the extracellular barriers are surpassed, there is an array of intracellular barriers inhibiting the effective functionality of miRNAs. Unlike DNA delivery wherein the plasmid has to be delivered to the nucleus, miRNA has an added advantage to be delivered to the cytoplasm [138]. However, there are numerous barriers to effective miRNA delivery. The first step is crossing the cell membrane to enter the cytoplasmic space of the cell [139]. For nucleic acid delivery, various physical methods such as electroporation, photoporation and sonoporation have been extensively evaluated and are successful in achieving intracellular delivery. However, these mechanisms result in a reduction of cell viability and, most importantly, cannot be used as an in vivo delivery strategy [140]. Nanoformulations generally rely on endocytosis as a mechanism to enter cells. Nevertheless, once the nanoformulations are engulfed through endocytosis, they mainly localize to early endosomes, which are then converted to late endosomes with a reduced pH and activation of degrading enzymes to eventually form lysosomes [141]. Thus, the nanoformulations that enter the cell through endocytosis need to escape the endosomes before lysosomal degradation. Endosomal escape is a major challenge in the field, and there is an extensive body of research to design polymers and lipids capable of achieving endosomal escape [141].

Many strategies have been evaluated to enable nanoformulations to undergo endosomal escape such as use of lysosomotropic agents [142], photosensitizers [143] and cell-penetrating peptides [144,145]. Additionally, multiple pore-forming and fusion peptides have been evaluated for their ability to promote the endosomal escape of the nanocarriers [146,147]. Studies have demonstrated the use of VIPER (virus-inspired polymer for endosomal release) to enhance siRNA and plasmid delivery [148]. Amphipathic polycations have also been evaluated as a strategy for endosomal escape [149,150]. Apart from all these strategies, we and many others have evaluated is the use of are polymers such as polyethyleneimine (PEI) or poly(amidoamine)s, which demonstrate osmotic swelling and electrostatic interaction with endosomal membrane for efficient gene delivery [151,152].

Hyaluronic acid-based nanoparticles for miRNA delivery to tumor microenvironment

We have improved the PEI with hyaluronic acid (HA), a natural polysaccharide formed of two disaccharide units of N-acetyl D glucosamine and D-glucuronic acid with β(1–4) interglycosidic linkage and is thus biodegradable [153]. Because HA is naturally present in the body, it does not elicit immune/inflammatory response and binds specifically to cell surface receptors such as CD44; thus, it can promote active cell targeting ability in the nanoformulation. Additionally, HA can be conjugated to polymers such as PEI by simple amide linkage using click chemistry. We have coupled HA to PEI in the presence of carbodiimide and N-hydroxysuccinimide to facilitate the amide linkage. The unreacted components are then removed by dialysis of the reaction solution using a 12- to 14-kDa MWCO dialysis membrane. The purified HA–PEI polymer is then lyophilized and stored at −20°C. The conjugation of HA with PEI is confirmed using ¹H-NMR spectroscopy (Figure 1A & B) [80]. Thus, HA is a widely used biomaterial in various delivery, therapeutic and theranostic applications [154–157]. HA conjugated with PEG has been used with other HA derivatives because PEG increases the circulation time and escapes the reticuloendothelial system [153,158]. Furthermore, these HA polymers have the ability to self-assemble when suspended in deionized water in the presence of nucleic acids (Figure 1C). PEI is positively charged and interacts with the negatively charged nucleic acids (miRNAs, in this case) through electrostatic interactions to form the core of the nanoparticles. The encapsulation of miRNAs with HA–PEI nanoparticles results in nanoparticles with hydrodynamic size ~100 nm as measured using a zetasizer based on dynamic light scattering (DLS) (Figure 1D). We further discuss these nanoformulations for delivery of miRNA-125b and miRNA-let-7b to the TME for overcoming immune suppression and for overcoming resistance to anticancer chemotherapeutics, respectively.

Figure 1. . Synthesis of hyaluronic acid derivatives and the formulation of microRNA-125b encapsulated nanoparticles.

(A) The ¹H NMR spectrum of the HA (20 kDa) and PEI shows the characteristic COCH₃ peak at ∽2 ppm for HA and peak for PEI between 2.5 and 3.0 ppm. (B) The ¹H NMR spectrum of the HA–PEI with carbodiimide and N-hydroxysuccinimide coupling does not show the PEI peak but shows additional peaks between 2 and 3 ppm due to PEI conjugation on the HA backbone. (C) The HA–PEI/miRNA particles made at 27:1 ratio in deionized water were visualized by transmission electron microscopy. Scale bar indicates 100 nm on both of the images. (D) Hydrodynamic diameters, PDI and ζ-potentials of HA–PEI and HA–PEI–miRNA-125b nanoparticle formulations. n = 4 identically prepared samples; data are expressed as mean ± standard deviation.

HA: Hyaluronic acid; NMR: Nuclear magnetic resonance; PDI: Polydispersity index; PEI: Polyethyleneimine.

Reproduced with permission from Parayath et al. [80].

Intricate mechanism of miRNA-125b in overcoming immune suppression

The implications of miRNA-125b in the TME have been widespread, affecting the phenotype of endothelial cells, immune cells as well as the growth and proliferation of cancer cells [159]. Within different cancer types, there is an extensive disparity related to expression levels of miRNA-125b. Overexpression of miRNA-125b is observed in cancers such as glioblastoma, retinoblastoma and acute myeloid leukemia. In contrast, in cancers such as hepatocellular cancer, melanoma, ovarian cancer, breast cancercinoma, colorectal carcinoma, osteosarcoma and endometrial cancer, there is clinical evidence of downregulation of miRNA-125b [159]. However, most cancers show dysregulation of miRNA-125b. This has necessitated a need for a deeper dive within cancer types to evaluate the mechanism of action of miRNA-125b in the TME. The role of miRNA-125b in inhibiting angiogenesis [160,161], programming tumor macrophages to proinflammatory phenotype [135] and activating immune responses [162,163] has been demonstrated. However, in contrast to the role of miRNA-125b in suppressing tumor growth and metastasis, studies have shown that expression of miR-125b in fibroblasts converts them to cancer-associated fibroblasts promoting tumor growth [164]. This illustrates the contrasting role of miRNA-125b in the TME and emphasizes the fact that miR-125b would play different roles in different cell types within the TME. Thus, it becomes essential to classify the role of miRNA-125b within different cell types of the TME.

Delivery of miRNA-125b to the TME in multiple cancer models

Reprogramming of TAMs to an antitumoral phenotype has been demonstrated as an effective strategy in cancer therapeutics. With the promising role of miRNA-125b in reprogramming TAMs, we and others have evaluated miRNA-125b as a cancer treatment strategy [80,84,165–167]. We evaluated the use of miRNA-125b for reprogramming TAMs to proinflammatory M1 phenotype in multiple cancer models. First, we evaluated the delivery of miR-125b in non-small-cell lung cancers because there is evidence of a positive correlation between M1 macrophage densities in the lung tissue and patient survival time [80]. We demonstrated macrophage-specific transfection using HA–PEI nanoparticles’ affinity for CD44 receptors on the macrophage cell surface. Furthermore, exploiting the ability of peritoneal macrophages to migrate toward the site of injury and/or inflammation, subsequently led to a >sixfold increase in the M1-to-M2 macrophage ratio in lung tissues from a non-small-cell lung cancer mouse model (Figure 2) [80].

Figure 2. . Repolarization of tumor-associated macrophages following intraperitoneal administration of miRNA-125b in hyaluronic acid–polyethyleneimine (HA–PEI) nanoparticle formulations in the KRAS/p53 genetically engineered non-small-cell lung tumor model.

(A) Lungs were isolated from mice on day 12, and immunohistochemistry was performed on lungs sections using CD11b, CD206 and CD80 antibodies. Representative images of random areas on each section were imaged using confocal microscopy. Scale bar = 20 μm. The ratio of relative mean fluorescence intensity of CD80/CD206 in the HA–PEI–miRNA-125b group was compared with the control and HA–PEI-scrambled miRNA group (n = 6); data are expressed as mean ± standard deviation; *p < 0.05 compared with control. (B) Cells from homogenized lungs were stained with F4/80 antibody for isolation using MACS magnetic beads, and RNA was extracted from these cells for expression of inducible nitric oxide synthase (iNOS) and Arg-1. The ratio of iNOS to Arg-1 in IL-4 stimulated peritoneal macrophages treated with HA–PEI nanoformulations. Quantitative PCR was used for quantification of the gene expression level with β-actin as the internal control.

HA-PEI: Hyaluronic acid–polyethyleneimine; miR = miRNA.

Reproduced with permission from Parayath et al. [80].

In the ovarian TME, CSF-1 stimulates macrophages to reprogram to the protumoral phenotype, further facilitating tumor growth [168]. Furthermore, in epithelial-derived ovarian cancers, there is a correlation observed between the macrophage number and the amount of CSF-1 increases with cancer growth [169]. Thus, our strategy was to reprogram TAMs from a protumoral to a proinflammatory phenotype using miRNA-125b as an anticancer therapy for ovarian cancers [84]. We have demonstrated the use of HA-based nanoparticles in the selective targeting and uptake by TAMs in the peritoneal cavity of a syngeneic ovarian cancer mouse model. Additionally, miRNA-125b encapsulated in HA–PEI nanoparticles could reprogram TAMs to an immune-promoting phenotype and can act as an adjuvant therapy along with paclitaxel, which is a standard of care for ovarian cancers (Figure 3) [84].

Figure 3. . Tumor-associated macrophage repolarization using miRNA-125b in HA–PEI nanoparticles.

Female C57BL/6 mice were injected with ID8 cells on day 1. On day 10, the mice were injected intraperitoneally with either HA–PEI-scrambled miRNA or HA–PEI–miRNA-125 b at 1 mg/kg dose on alternate day for 3 days, and the peritoneal cavity was lavaged with 1× phosphate-buffered saline (PBS) on day 16, for peritoneal cells collection and analysis by FACS for (A) ratio of CD206 to CD80. Expression of (B) inducible nitric oxide synthase (iNOS)/Arg-1 ratio tumor-associated macrophages treated with HA–PEI nanoparticle formulations. Quantitative PCR was used for quantification of gene expression level with β-actin as the internal control. (C) Female C57BL/6 mice were injected with ID8 cells on day 1. On day 8, mice were injected intraperitoneally with paclitaxel (2 mg/kg), and on day, 10 mice were injected intraperitoneally with either HA–PEI-scrambled miRNA or HA–PEI–miRNA-125 b at 1 mg/kg dose on alternate days for 3 days, and peritoneal cavity was lavaged with 1× PBS on days 16 and 24. VEGF levels were determined by ELISA in the peritoneal lavage from mice. N = 4; data are analyzed by one-way analysis of variance.

HA: Hyaluronic acid; miR: miRNA; ns: No significance; PEI: Polyethyleneimine.

Reproduced with permission from Parayath et al. [84].

TAMs play an important role in tumor growth in pancreatic ductal adenocarcinoma (PDAC). The complicated TME has been an obstacle to effective PDAC therapy. PDAC progression shows an inverse correlation with numbers of TAMs in pancreatic cancer tissue [170,171], which validates reprogramming of TAMs as an effective therapeutic strategy for fast-growing cancers such as PDAC. In a genetically engineered rodent model of PDAC, we demonstrated preferential accumulation of miRNA-125b encapsulated HA-based nanoparticles encapsulating miRNA-125b in the pancreas of PDAC mice [165]. Moreover, we employed an additional mechanism to target M2 TAMs specifically by conjugating the M2 peptide to HA–PEI nanoparticles [165]. These miRNA-125b encapsulating nanoparticles effectively reprogrammed the TAMs in PDAC to M1 phenotype (proinflammatory) and thus can be evaluated further to understand its potential as an anticancer therapeutic in the PADC model.

miRNA-let-7b therapy to overcome chemoresistance

The aberrant expression of miRNA-let-7b has been correlated with cancer growth and development [172]. The miRNA-let-7 family members have sequence similarity and thus have similar functions [173]. The targets of let-7 targets are oncogenes and other genes critical for cancer growth such as Myc, RAS, HMGA2, VEGF, Lin28 MDR family and long noncoding RNA H19 [174]. The decreased expression of let-7b expression is correlated with ovarian cancer growth and poor prognosis. let-7b levels were shown to be influenced by the two major subtypes: the let-7b high- and let-7b low-expressing tumor cells. Monitoring the levels of let-7 after surgery and chemotherapy has demonstrated its utility as a biomarker. Reduced expression of let-7 is linked to fast-growing cancer with poor prognosis. Similarly, increased let-7 levels are associated with increased lifespan and better prognosis [175–177]. The expression of levels of let-7 have also been shown to differ based on the stage and site of the tumor; for example, miRNA-let-7 shows low expression at metastatic sites compared with the primary cancers [178–180]. Overall, all the aforementioned studies highlight the role of let-7 family members as a biomarker for advanced-stage cancer.

The multidrug resistance (MDR1) protein has been associated with developing resistance to therapeutic drugs [181]. let-7d has been shown to modulate MDR1 in patients treated with chemotherapy after surgical treatment, and loss of let-7d provides a suitable microenvironment for the development of resistance to chemotherapeutics [182]. Thus, there is a good correlation between lowered levels of let-7d and IMP-1 and MDR1 proteins in patients with relapsed ovarian cancer after chemotherapy. Several studies have shown that induction of let-7 expression modulates chemosensitivity in cancer cells and cancer mice models due to inhibition of chemoresistance genes and LIN28A/B, STAT3, E2F1 and IMP1 [174]. Let-7 overexpression in mice model studies show tumor regression and improved survival [183–185]. All these studies led us to hypothesize that delivery of let-7b to the tumor tissue would help protect against recurring chemoresistant ovarian carcinoma.

Delivery of miRNA-let-7b in a relapsed or resistant ovarian cancer model

Because delivery of miRNAs to tumor cells in vivo is challenging, we and others have evaluated the potential of HA–PEI polymers to deliver miRNAs [145]. The cotreatment of HA–PEI–let-7b nanoparticles and paclitaxel has resulted in reduced IC50 values for paclitaxel in resistant ID8 ovarian cancer cells. Furthermore, we observed 3.5-fold reduced expression levels of miRNA-let7b levels in chemoresistant mice model of ovarian cancer compared to expression levels of miR-let7b levels in naïve mice. Additionally, expression levels of miRNA-let-7b are back to baseline in mice treated with HA–PEI encapsulating miR-Let7b nanoparticles (Figure 4A) in a chemoresistant mice model of ovarian carcinoma. Furthermore, we observed a 30–70% reduction in mRNA levels of eight oncogenes in tumors of ovarian cancer mice compared with control (Figure 4B). Additionally, our study demonstrated lowered VEGF levels (20- and 10-fold) in dual paclitaxel and HA–PEI–miRNA-let-7b treated group compared with the control and paclitaxel or HA–PEI–let-7b individual treatment groups respectively [98]. Furthermore, delivering additional miRNAs that show lower expression in tumors would potentially help further improve efficacy. This needs to be validated in appropriate cancer models.

Figure 4. . Treatment of relapsed and refractory ID8-VEGF ovarian cancer bearing mice with microRNA Let7b.

(A) Quantification of absolute copies of let-7b in tumor nodules 48 h after three doses of let-7b dosing (intraperitoneal) (n = 4 per group). (B) Relative quantification of let-7b mediated target gene expression in changes 48 h after three doses of HA–PEI–let-7b/scr (n = 4 per group). (C) Measurement of antitumor efficacy of combination therapy using VEGF ELISA. VEGF levels (pg/ml) were measured from supernatants of ascitic fluid on day 55 (n = 6–8 per group). (D) Representative images of ID8-VEGF resistant tumor-bearing mice on day 55 after tumor inoculation and untreated control (resistant/relapse) showing ascitic fluid buildup, leading to abdominal bloating, paclitaxel (PTX) only with ascites in the peritoneal cavity and PTX + HA–PEI–let-7b group with no ascites formation (p-values for A, B and C = ****p < 0.0001; ***p < 0.001; **p < 0.01, respectively, and were obtained by Student t-test comparing PTX + HA–PEI–let-7b with the other groups).

HA-PEI: Hyaluronic acid–polyethyleneimine; PTX: Paclitaxel.

Conclusion

miRNAs are important players in cell-to-cell communications in the tumor microenvironment and are known to modulate the tumor immune landscape. Inducing favorable miRNAs into the tumor microenvironment to hinder tumor development by activating intrinsic immune cells of the tumor microenvironment is an encouraging anticancer strategy. Delivering miRNAs to the tumor microenvironment to reprogram the TAMs to perform antitumor functions has been an efficient strategy to stimulate the immune system and can be exploited as a combination therapy with the current standard of care cancer treatment options. Furthermore, another class of miRNAs which are the tumor suppressors miRNAs have shown promising results in overcoming chemoresistance. With the increasing acceptance of the nonviral delivery systems for delivering nucleic acids and the advancement of tools for miR profiling of tumors, it is essential to extensively investigate the therapeutic potential of miRNAs as a cancer treatment strategy as monotherapy or adjuvant therapy.

Future perspective

Numerous miRNAs have been identified to overcome immunosuppression and chemoresistance in various cancer models. For effective delivery of these miRNAs to the TME, a competent delivery system is the key criterion for the success. With the rapid advancement of the field of delivery systems and the success of lipid nanoparticles for the COVID vaccine, the delivery of tumor-targeted delivery miRNAs could be the next step in the clinic. However, an important factor to consider with miRNAs is the target specificity. Because these small oligos could potentially have multiple targets, it could be important to consider the off-target effects of these miRNAs in the TME. Additionally, the kinetics of action of these miRNAs as the adjuvant therapy to other anticancer therapies needs to be evaluated in depth. Nonetheless, there will be a surge in RNA therapeutics in clinical settings, and miRNAs could certainly be an important part of these therapeutic modalities.

Executive summary.

• The tumor microenvironment is a complex environment comprised of varied cellular and non-cellular components that interact with one another through messenger molecules to facilitate tumor growth and progression.

• MiRs are a class of small non-coding RNAs that play a vital role in cell-to-cell communication within the tumor microenvironment.

• MiR profiling of the tumor microenvironment has revealed the importance of miRs in determining the immune landscape of the tumors and the probability of tumors developing chemoresistance.

• Self-assembled nanoparticles of hyaluronic acid (HA) modified with polyethyleneimine (PEI) are an efficient tool to deliver the miRs to the specific cell type within the tumor microenvironment.

• HA–PEI nanoparticles can deliver miR-125b to tumor-associated macrophages and reprogram these macrophages to antitumoral phenotype and can act as an adjuvant therapy along with paclitaxel for ovarian cancers.

• Combination therapy of paclitaxel and HA–PEI encapsulating miR-let7b shows a significant reduction in tumor growth in a relapsed mice model of ovarian cancer.

• The ability to modulate the miRs of the tumor microenvironment in order to overcome immunosuppression and therapeutic resistance is a promising anticancer treatment strategy.