Development of siRNA Delivery System by Lipid Nanoparticles Modified with Functional Materials for Cancer Treatment

Abstract

Nucleic acid drugs can control gene expression and function in a manner different from that of conventional compounds. On the other hand, nucleic acids can be easily degraded in the in vivo circumstances. In addition, nucleic acids cannot penetrate cell membranes. Therefore, a drug delivery system (DDS) is essential to protect nucleic acid molecules until they reach the target cell and to release them efficiently inside the cell. In order to apply nucleic acid drugs to new cancer therapeutic strategies, the author has been developing a DDS that enables functional control of vascular endothelial cells that consist of the tumor microenvironment. The aim of my study is to develop lipid nanoparticles (LNPs) were modified with functional molecules that control their pharmacokinetics in vivo and intracellular fate to delivered small interfering RNA (siRNA) to tumor vasculature. By imparting pH-responsive membrane fusion properties to lipid nanoparticles, I have developed a system that responds to acidification in endosomes within cells and subsequently efficiently releases siRNA into the cytoplasm via membrane fusion, where siRNA molecules exhibit their function. In addition, by developing a method for presenting functional molecules, such as peptides, saccharides and so on, that recognize target cells on the surface of LNPs, I succeeded in establishing LNPs which internalize more efficiently into specific cells than off-target cells. Finally, by integrating these technologies, I developed an in vivo siRNA DDS that enables in vivo control of genes of interest in tumor vascular endothelial cells and succeeded in cancer therapy by regulating vascular function.

1. PROS AND CONS: NUCLEIC ACIDS DELIVERY

Nucleic acids have been a principal subject of much research in the field of drug development because of their ability to control biological functions through a mechanism of action that is completely different from that of conventional low-molecular-weight compounds and protein-based drugs; the target of nucleic acids is transcriptomic RNA and genomic DNA. Nucleic acid medicine began with plasmid DNA that was biologically generated in a procaryote Escherichia coli, which can be used to produce proteins of interest. Since then, tremendous research on nucleic acid medicines have been conducted on new modalities, such as small interfering RNA (siRNA) and other small synthetic nucleic acids, as well as mRNA. Among them, it is particularly noteworthy that siRNA has attracted considerable interest as a new type of nucleic acid medicine that can cause RNA interference with short length RNA. This approach was discovered by Drs. Fire and Mello, who received the Nobel Prize in Medicine in 2006.¹⁾ This is because, unlike conventional low-molecular-weight compounds, siRNAs can be designed against any gene based on the identification of a transcriptome sequences accompanied by progress in DNA sequencing technologies.

Nucleic acids are thus very useful molecules, but they are easily inactivated by nucleic acid-degrading enzymes that are abundant in body fluids, which motivate us to develop a drug delivery system (DDS) for their delivery to the target cells an essential part of this process. Various DDSs have been developed for delivering siRNAs, including polymeric polymers and inorganic compounds.^2,3) The most advanced DDS is the lipid nanoparticle (LNP), the structure of which is like liposomes in terms of nanoparticle composition, but it has a different nano-structure.^4,5) Liposomes are nano-sized vesicles with an aqueous phase inside, whereas lipid nanoparticles are generally packed with higher-order structures of nucleic acids and lipid molecules, although there is room for debate, and there is no clear aqueous phase inside.⁶⁾ Onpattro^® is the first-in-human siRNA medicine using an LNP formulation that has been approved to use. Onpattro^® was approved by the food and drug administration in 2018 for the treatment of familial amyloid neuropathy.⁷⁾ Until the introduction of Onpattro^®, the disease had only been treated symptomatically, but Onpattro^® has made it possible to delay the progression of the disease itself by inhibiting the production of transthyretin in hepatocytes, the deposition of which is a cause of neuropathy. The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (CoV-SARS-2) also greatly accelerated the development of RNA drugs.⁸⁾ As many readers may remember, the vaccines against CoV-SARS-2 are LNP formulations containing mRNA that encodes for the spike protein as an antigen. The details of these LNP drugs are beyond the scope of this article, but LNP-based nucleic acid drugs are now one of the more important platforms in clinical practice. On the other hand, the target cells of LNPs are currently limited to hepatocytes, which have an endogenous mechanism of that permits LNPs to be taken up in vivo, or to cells with phagocytic ability that can take up nanoparticle-based formulations.⁹⁾ Therefore, the development of a cell-selective LNP technology would contribute to the further development of LNP drug discovery. Based on the above background, I focused my research on methodology for controlling the localization of nucleic acids, especially regarding on their distribution in the body.

2. TARGETING TUMOR MICROENVIRONMENT

In 1986, Drs. Matsumura and Maeda developed a process that permitted macromolecules with a prolonged circulation time to passively accumulate in tumor tissue.¹⁰⁾ Since this enhanced permeability and retention (EPR) effect was proposed, research has focused on the development of tumor-targeted DDS, especially using nanoparticles with blood retention properties.^11,12) Concerning lipid-based DDS, it was discovered that modifying the surface of liposomes with polyethylene glycol (PEG) can result in a sufficient blood retention for tumor delivery,^13,14) leading to the development of doxorubicin-encapsulated PEGylated liposomes, Doxil^®.¹⁵⁾ Although Doxil^® is widely used in the treatment of some types of cancer, a meta-analysis of the pharmacological effects of liposomal formulations in clinical and non-clinical settings revealed that the benefits of nanoparticle formulations were not as apparent in the clinic as was originally anticipated in 2016.¹⁶⁾ Thus, in recent years, the effectiveness of strategies that target cancer cells themselves through EPR effects has been questioned.^17,18) Various reasons have been proposed for this, one of which is that clinical tumor tissue has a more rich stroma than non-clinical models, and consequently nanoparticles can diffuse within the perivascular space, indicating that nanoparticles that encapsulate anti-cancer drugs do not reach cancer cells. Therefore, I conceived of developing a DDS that does not target cancer cells themselves.

Tumor tissues form a cancer microenvironment in which various types of non-cancer cells around the cancer cells promote their growth. Tumor microenvironment has recently attracted much attention as a new target for cancer drug discovery, such as cancer-associated fibroblasts and cancer-associated macrophages.^19,20) My research focused on vascular endothelial cells. When cancer cells grow to a size of more than a few mm³, they inevitably grow new blood vessels to supply oxygen and nutrients. This was originally proposed by the late Dr. Folkman.²¹⁾ Since the identification of vascular endothelial cell growth factor (VEGF) as a factor that promotes angiogenesis,^22,23) the development of inhibitors of VEGF and its receptors has progressed, and the anti-VEGF antibody Avastin^® was developed as the result of this research. Avastin^® is now used as a drug. Although anti-VEGF therapy has shown good therapeutic results, side effects on normal tissues other than tumor tissue is a problem.^24,25) Based on the above discussion, I attempted to establish an siRNA delivery system that targets cancer neovascularization and my goal is to create a new type of cancer therapy that does not target cancer cells themselves.

3. REGULATING THE INTRACELLULAR TRAFFICKING OF SIRNA

In order to target cancer neovascularization with siRNAs, intracellular dynamics, i.e., the “intracellular fate” of the siRNA from the site of administration to the blood vessels, and the “intracellular fate” of the siRNA from the endosome to the cytoplasm after it is taken up by the target cells must be skillfully controlled. To achieve this, I constructed a smart DDS which allows their cargo to overcome various biological barriers via the incorporation of spatio-temporally arranged functional devices.

I initially worked on a method to control the intracellular dynamics of siRNAs. siRNAs are taken up by cells by endocytosis, then incorporated into vesicles called endosomes, and are finally transferred to organelles called lysosomes, where they undergo degradation. Therefore, in order for siRNA to exhibit its function properly, it must not be taken up by endosomes before migrating to lysosomes. I focused on the acidification of endosomes as they migrate to lysosomes and designed a functional element that exhibits membrane fusion function in response to acidification (Fig. 1). After influenza viruses enter cells during an infection, hemagglutinin on the viral surface undergoes a conformational change in response to endosomal acidification, inducing membrane fusion which allows it to escape into the cytoplasm. The GALA peptide, designed in homage to this viral entry mechanism, is an artificial peptide that contains four glutamate-alanine-leucine-alanine repeats and undergoes a conformational change to a structure that is rich in hydrophobic-rich α-helices upon acidification.²⁶⁾ When a cholesterol derivative of this GALA peptide was located on the surface of the LNP; the GALA-modified LNPs showed a significantly enhanced activity in vitro.²⁷⁾ On the other hand, because this GALA peptide is a long chain peptide consisting of 30 amino acids, LNPs that are modified with this cholesterol GALA peptide are recognized by immune cells in vivo and are rapidly cleared from the circulation. Therefore, I truncated the GALA sequences so that it has both biocompatibility and fusogenic activity by reducing the number of GALA repeats to improve biocompatibility. The resulting truncated GALA peptide (shGALA)-modified LNPs showed a blood retention that was comparable to that of unmodified LNPs, and this allowed the siRNA to be delivered to cancer tissue.²⁸⁾

Fig. 1. Illustrations of Functional Molecules That Induce Membrane Fusion and/or Destruction in Response to Lowered pH in Endosomes

In order to confer pH-dependent membrane fusion activity on the lipid molecule itself; Dr. Sato et al. developed the YSK series of pH-responsive cationic lipids that contain a tertiary amino group with a pK_a of around 6 as the hydrophilic group.²⁹⁾ Among these cationic lipids, he incorporated YSK05, which has the best in vitro activity, into LNPs (YSK-LNPs) for siRNA delivery to cancer tissues. I then applied the stem-loop PCR method for the quantification of micro RNA to quantify the amount of siRNA delivered to tumor tissues. The results showed that the PEGylated YSK-LNPs transferred the same amount of siRNAs to cancer tissues as lipids. Furthermore, a dose of 3.0 mg/kg induced the suppression of gene expression at both the mRNA and protein levels.³⁰⁾ Since it has been reported that siRNAs suppress gene expression through non-specific effects that differ from the original RNA interference mechanism, I performed the rapid amplification of the 5′ cDNA ends (the 5′ RACE) method to detect the cleaved mRNA by the cDNA. Unlike the conventional quantitative reverse transcription-PCR method in which the remaining amount of mRNA is measured, this is a head-to-head method that detects the cleaved mRNA and the cleaved mRNA at the cleavage site predicted by the mechanism of RNA interference, and is the most effective method for achieving the desired effect of siRNA. This is one of these methods. As a result, when the YSK-LNP was administered, the mRNA obtained by the transfected siRNA was indeed detected. In conclusion, I was able to demonstrate that regulating intracellular dynamics by pH-dependent membrane fusion activity by YSK05 is a useful method that can actually exhibit function in vivo.³¹⁾

4. TARGETING THE TUMOR VASCULATURE USING A TARGETED LNP

Having established that the siRNA DDS can be adapted in vivo, the next step was to develop a technology for controlling the pharmacokinetics, i.e., to impart cell homing ability to the LNP. I developed a method to maximize the activity of functional elements such as peptides and polysaccharides on the lipid membrane surface of the YSK-LNP by synthesizing new lipid derivatives and devising additives during LNP production.^32–34) Based on these techniques, I designed RGD-LNPs, i.e., LNPs that are modified with cyclic RGD peptides that have affinity for the αVβ3 integrin dimer, which is highly expressed in tumor endothelial cells of the tumor vasculature. The RGD-LNP would be expected to recognize αVβ3 integrin dimer on tumor endothelial cells, and then be internalized into cells where they would release siRNA into the cytosol (Fig. 2a). As a result of optimizing the lipid composition and peptide modification conditions, the RGD-LNP showed a 50% suppression of tumor vascular endothelial markers^35,36) (Fig. 2b). This was the highest-performing DDS in the world at the time for a cancer vascular selective system. In addition, observations of the intra-tumor localization of the RGD-LNP after intravenous administration showed a perfect match with the tumor vasculature. Furthermore, mRNAs of tumor vascular endothelial markers that are cleaved by the 5′ RACE-PCR method described above were detected, indicating that the RGD-LNP is indeed capable of delivering siRNA to tumor vascular endothelial cells and inducing sequence-specific silencing. In addition, to demonstrate the therapeutic potential of delivering siRNA to cancer vessels, LNPs containing siRNA against the VEGF receptor 2 (VEGFR2), the major VEGF receptor in tumor endothelial cells, were administered, and a strong inhibition of cancer growth was induced.³⁵⁾ In conclusion, my research has demonstrated the development of siRNA-based cancer microenvironment-targeted DDSs and their therapeutic effects. In addition, one of the characteristics of siRNA is that it is possible to suppress the function of a specific gene by simply changing the sequence of the siRNA that encapsulates the function of the desired gene, since the entire genome sequence has recently been revealed. Based on their own genetic screening of cancer vascular endothelial cells, Dr. Hida and his colleagues at the Graduate School of Dentistry, Hokkaido University, identified Biglycan as an angiogenesis-promoting gene that is present in blood vessels in tumor tissue but not in normal tissue. They found that RGD-LNPs containing siRNA against Biglycan and administered to tumor-bearing cancer mice improved the abnormal vascularity of tumors and inhibited cancer growth.³⁷⁾

Fig. 2. The in Vivo Gene Silencing Effect on Tumor Endothelial Cells by RGD-LNP

(a) Schematic diagrams showing a series of processes from recognition of tumor endothelial cells to siRNA release. (b) The in vivo gene silencing effect by RGD-LNP. The RGD-LNP preparation was administered into tumor-bearing mice at a dose of 0.75–3.0 mg siRNA/kg bodyweight. The Y-axis indicates gene expression percentage to non-treatment group. The data was cited by ref. No. 36.

5. COMBINATION THERAPY OF CONTROL OF TUMOR MICROENVIRONMENT AND NANOTHERAPEUTICS

The conventional EPR effect lacks the endothelial cell-to-cell adhesion and lining cells/proteins that tumor vessels would normally have due to their rapid growth rate. This has been attributed to increased leakage contributing to the accumulation of nano-sized molecules. In fact, there have been reports of further enhanced nanoparticle migration based on innovations that further improve vascular permeability. However, this does not necessarily have a positive effect on the accumulation of nanoparticles, such as the enhancement of excessive angiogenesis or fibrosis that is induced by inadequate blood flow and the resulting hypoxia (Fig. 3a). Based on these facts, I hypothesized that “vascular normalization,” which suppresses angiogenesis, would improve nanoparticle migration, especially in certain types of cancer where angiogenesis is naturally active. Based on this hypothesis, I developed a strategy for improving the migration of nanoparticles into cancer tissues by suppressing VEGFR2 in renal cell carcinoma, where angiogenesis is active. At a dose of 3.0 mg/kg, the amount of liposome formulation subsequently administered could be increased significantly, as was its diffusion within the tumor tissue³⁸⁾ (Fig. 3b). The percentage of blood vessels with blood flow in the tumor tissue was also increased, suggesting that the number of dysfunctional vessels had been reduced, and blood flow was restored. In addition, the levels of collagen type I, an extracellular matrix molecule in the tumor tissue, decreased and this decrease was inversely proportional to the dose, suggesting that extracellular matrix (ECM) remodeling occurred due to the resolution of hypoxia. These decreases were accompanied by an increase in the production of M1 macrophages in the tumor tissue, and the removal of these macrophages by clodronate liposomes cancelled this improvement in liposome drug migration. In addition, the administration of an inhibitor of matrix metalloproteinase, which is produced by macrophages and may contribute to extracellular matrix degradation, also cancelled the improved liposome diffusion. In conclusion, my findings indicate that the administration of RGD-LNPs induces vascular normalization, which stimulated the production of perivascular macrophages and extracellular matrix remodeling, resulting in a dramatic improvement in the migration of nanoparticle formulations into tumor tissues.³⁹⁾ Finally, the therapeutic efficacy of a combination therapy with doxorubicin-encapsulated liposomes and RGD-LNPs as liposome formulations was verified. The renal cell carcinoma used in this study was resistant to low concentrations of doxorubicin, and doxorubicin-encapsulated liposomes (DOX-liposome) showed no significant therapeutic effect. On the other hand, the use of a combination of RGD-LNPs and doxorubicin-encapsulated liposomes showed a significant therapeutic effect compared to the untreated group (Fig. 3c). On the other hand, I was able to elucidate the mechanism by which the normalization of blood vessels by the RGD-LNP occurs, which implies a decrease in leakiness, paradoxically, an improved migration of liposomal formulations. When the RGD-LNP simultaneously suppressed VEGFR2 and inhibited Caveolin-1, the improvement in volume was found to be cancelled.⁴⁰⁾ Therefore, it is possible that caveolae-dependent transcytosis may contribute to the migration from blood vessels normalized by RGD-LNP to tumor tissue. One of my objectives is to further examine this possibility in the future.

Fig. 3. A New Strategy Which Involves Vascular Normalization by siRNA Delivery to Tumor Endothelial Cells and Nanoparticles Targeting Cancer Cells

(a) The concept of improving the intratumor distribution of nanoparticles in combination with tumor vasculature normalization. (b) Enhanced diffusion of liposomes by vascular normalization. The Y-axis indicate liposome signal intensities normalized to the brightest signals from the tumor vasculature to a deeper area. (c) Therapeutic effect by the combination therapy. Tumor-bearing mice were administered the DOX-liposomes and/or RGD-LNP encapsulating siRNA against VEGFR2. Tumor progression was synergistically suppressed by the combination therapy. **: p < 0.01 (SNK test, n = 3–5). These data were cited by ref. No. 38 and slightly modified.

The study is supported, in part, by the Japan Society for the Promotion of Science for Young Scientist [18K18351, 20K20195], by the Mochida Memorial Foundation, the Takeda Memorial Foundation and the Terumo Life Science Foundation.

Acknowledgments

I wish to acknowledge Prof. Hideyoshi Harashima (Hokkaido University), Prof. Hidetaka Akita (Tohoku University) and Prof. Hiroto Hatakeyama (Chiba University). I am most grateful for kind cooperation by all of the laboratory members at the Laboratory of Molecular Design of Pharmaceutics and the Laboratory of Innovative Nanomedicine in Hokkaido University.

Conflict of Interest

The author declares no conflict of interest.

Notes

This review of the author’s work was written by the author upon receiving the 2022 Pharmaceutical Society of Japan Award for Young Scientists.

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