MicroRNA (miRNA) in Nanostructure Lipid Carriers based Tumor Gene Therapy

Introduction

MicroRNAs is a non-coded double stranded RNAs made of approximately of 20 nucleotides disclosed in 1993 by Ambros and Ruvkun laboratories that has significant role in regulation of gene expression at post transcriptional level [1–2]. It has been identified that the non-coding region of mRNA is capable of providing targeting signals in RNA binding protein domain. The all-embracing research studies directed that miRNA has important contribution in cell proliferation, migration metastasis and development of tumor. Thus, miRNA has been evolved as a novel therapeutic target in cancer therapy [3-4]. NLC is second generation solid lipid nanoparticle comprised of solid lipid mixed with liquid lipid (oil) which is advancement in solid lipid nanoparticles. NLC as nanocarrier could able to provide controlled release of drug candidate and has been widely adopted as drug delivery carrier for tumor targeting. The potential capability of NLCs to traverse across leaky vasculature and passively accumulated in neighboring tumor cells, and tissues (EPR effect) make them ideal candidate for tumor gene therapy. However, the surface functionalisation of NLC with protein, ligand, antibodies, aptamers, and miRNA further warranted a novel promising therapeutic strategy in tumor therapy [5]. Scientist has tried various ways of potential delivery of miRNA into cells employing different novel carriers including NLCs which has achieved significant advance in miRNA delivery [6]. The aqueous core in NLCs is surrounded by a lipid bilayer and achieved high encapsulation efficiency, payload, and biostability [7]. Among the three categories of NLCs surface functionalized NLCs has been widely explored for nucleic acid delivery along with miRNA for tumor gene therapy owing to low immunogenicity, biocompatibility, high biodegradability etc and expected that has undimmed clinical feature ahead [7].

Surface functionalised NLCs in mRNA delivery

The passively targeted NLCs are easily recognised and undergo phagocytosis by from endo-reticular or macrophagic system and finally cleared out from the body. Thence, to prevent phagocytosis by macrophage and enhance retention inside body NLCs could be surface modified by coating with biocompatible polymers such as PEG which enables to enhance circulation time in blood stream thereby increase biological half life. Further tissue specific and selective ligated NLCs could enable to enhance concentration of miRNA/NLC complex in the target tissue in vivo.

In an attempt to enhance miRNA delivery utilising NLCs Hayward and associates modified the surface of NLCs using hyaluronic acid with intention to improve targeting capability, life span and cell permeability in HER2 positive metastatic breast cancer. The investigation has shown that miRNA-125-a-5p principally targeted metastatic breast cancer cells which was isolated from endocytosis, and finally escapes from endo-lysosomal pathway for inducing effective gene silencing, and further knock out proto-oncogenes (HER2) involved in post transcriptional and translational regulations of gene. At the same time, related molecular pathway including the RAS/RAF/ERK/MEK/MAPK signaling and PI3K/Akt/mTOR pathways, proliferation, migration and metastasis were also reduced significantly.

The ligand tagged (transferrin) NLCs specially designed by Zhang and coworkers for the delivery of miR-221 antisense oligonucleotides (anti-miR-221) to hepatocellular carcinoma (HepG2) cell line. The outcomes of the study indicated average size of particles were 122.5 nm and encapsulation efficiency was 70 %. Further, transferrin tagged NLCs was stable at 4 oC and delivered anti-miR-221 more efficiently and led to ameliorated efficacy compared to  nontargeted NLCs in the HepG2 cells through receptor mediated endocytosis process [8].

In the similar context Chen and associates [9] accounted the miR-34a delivery through functionalised NLCs with single-chain variable fragment (scFv) for tumor-targeting for the treatment of metastatic melanoma cells. The experimental results demonstrated that miR-34a was efficiently delivered by scFv targeted NLCs in induced tumor and caused cell apoptosis and further oppressed cell migration in vivo which was assorted with suppression of MAPK signaling and down regulation of survivin. Recently, other works supported the efficient delivery of functionalised target oriented NLCs largely depends upon the particle size and entrapment efficiency of therapeutics. For instance, Lee and colleagues [10] documented that surface modified NLCs binds with receptor 1 ephrin A-type were used to deliver let-7-a into non-small cell lung cancer (NSCLC) of mouse models. The findings showed that the ephrin-A1 modified NLCs of average diameter ~100 nm exhibited high payload, stability, and low cytotoxicity of ephrin-A1 and let-7a. In addition, ephrin-A1 altered NLCs could suppress NSCLC proliferation, cell migration and tumor growth, besides improving the effective delivery of let-7a.

Moreover, the temperature of tumor cells and neighboring area play significant role in delivering the active content from functionalised NLCs along with limited particle size, and high payload and relationship of tumor cell temperature and effective delivery in vivo remains to be sorted out in future. The combinatorial targeted delivery of functionalised NLCs has also achieved progress. In an attempt to treat brain tumor Costa and coworkers [11] designed chlorotoxin (CTX) targeted NLCs with payload of anti-miR-21 for therapeutic intervention in brain tumor. The results showed that the NLCs achieved more than 85 % of entrapment efficiency of anti-miR-21 with reported mean particle size~190 nm. Further experiment led to preferential accumulation of anti-miR-21 by the CTX targeted NLCs within brain tumor upon i.v. administration with no indication of systemic immune injury. Moreover, the combinatorial systemic delivery of sunitinib with anti-miR-21 delivered by CTX targeted NLCs demonstrated  potential inhibition of proliferation, enhanced apoptosis and thereby increased mean survival of GBM-bearing mice. The study indicated that potential combinatorial therapeutic strategy for the delivery of anti-miR-21 through surface modified NLCs.

Positively charge NLCs in mRNA delivery

The positively charge or cationic NLCs can be employed as a carrier for negatively charged active substance delivery such as DNAs, RNAs, polypeptides, proteins, and oligonucleotides. The positively charged NLCs comprised of (a) a cationic head, (b) hydrophobic skeleton or backbone and (c) a linker. The positive charge in this type NLCs have significant role in potential delivery of miRNA due to surface charge interaction. For example, Chen and colleagues [9] observed that positively charge NLCs have been prosperously delivered miR-34a in murine model of lungs metastatic melanoma. Post treatment tumor cell migration suppressed effectively in vivo, suggesting that cationic NLCs efficiently delivered miRNAs in vivo. It is further reported that the head and neck squamous cell carcinoma has been treated with positively charge NLCs through delivery of deliver miR-107 both in vitro and in vivo.

Neutral NLCs in miRNAs delivery

Currently, neutral NLCs have paid wider attention as a novel drug carrier for the delivery of miRNAs in tumor gene therapy. The neutral NLCs have advantages of charge less and don’t form aggregates in biological fluids and not easily cleared by liver and macrophagic system [12]. For example Trang and associates [13] reported miR-124 delivery neutral NLCs through tail-vein injection in mouse lung cancer model. Post injection analysis was carried on both the blood and different organs which indicated that elevated level of miR-124 from neutral NLC complex. Further study established that significant amount of miR-124 was ingested by cells or tissues in blood and organs in mice as confirmed by 0.9 % saline perfusion analysis. It is worth mentioning that 0.9 % saline perfusion study revealed reduced level of miR-124 by 70–80 % in vital organs viz., kidney and liver. Literature further cited some reports on miR-34a and miR-495delivery by neutral NLCs in lung cancer cell achieved good therapeutic efficacy [14].

Conclusion

The aforementioned study concluded that the current application of NLCs as nanocarrier looks promising and better future for target oriented delivery of miRNAs in tumor gene therapy as well as combinatorial therapy. Moreover, the close examination of particle size, effective delivery of payload could further be sorted out the challenges involved in process of tumor gene therapy in vivo. The surface modified NLCs and their size constraint matters for receptor oriented targeting for efficient delivery miRNA and cellular uptake, organ and tissue ingestion in vivo should be evaluated prospectively. Additionally, the pharmacokinetic, release mechanism, in-depth study of molecular structures of NLCs will further provide insight into a better mechanistic understanding in tumorigenesis.

References

1. Md. Habban Akhter, Md. Shamshir Alam, Md. Akram Minhaj. Smart Nano-Enabled Drug Carrier in Combating Tumor Development and Progress.
https://www.pharmafocusasia.com/articles/smart-nano-enabled-drug-carrier-in-combating-tumor-development.
2. Gabriele Varani. Twenty years of RNA: the discovery of MicroRNAs. https://rnajournal.cshlp.org/content/21/4/751.full.
3.  Weizi Hu, Tan Chunli, He Yunjie. Functional miRNAs in breast cancer drug resistance. Onco Targets Ther. 2018;11:1529–41.
4. Zheng L, Chen J, Zhou Z, et al. miR-195 enhances the radiosensitivity of colorectal cancer cells by suppressing CARM1. Onco Targets Ther. 2017;10:1027–38.
5. Akhter MH, Rizwanullah M, Ahmad J, et al. Nanocarriers in advanced drug targeting:
setting novel paradigm in cancer therapeutics. Artif Cells Nanomed Biotechnol 2017;
http://dx.doi.org/10.1080/21691401.2017.1366333.
6. Akinc A, Zumbuehl A, Goldberg M, et al. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat Biotechnol. 2008;26(5):561–9.
7. Mokhtarieh AA, Lee J, Kim S, et al. Preparation of siRNA encapsulated nanoliposomes suitable for siRNA delivery by simply discontinuous mixing. Biochim Biophys Acta. 2018;1860(6):1318–25.
8. Zhang W, Peng F, Zhou T, et al. Targeted delivery of chemically modified anti-miR-221 to hepatocellular carcinoma with negatively charged liposomes. Int J Nanomed. 2015;10:4825–36.
9. Chen Y, Zhu X, Zhang X, et al. Nanoparticles modified with tumortargeting scFv deliver siRNA and miRNA for cancer therapy. Mol Ther. 2010;18(9):1650–6.
10. Lee HY, Mohammed KA, Kaye F, et al. Targeted delivery of let-7a microRNA encapsulated ephrin-A1 conjugated liposomal nanoparticles inhibit tumor growth in lung cancer. Int J Nanomed. 2013;8:4481–94.
11. Costa PM, Cardoso AL, Cunha P, et al. MiRNA-21 silencing mediated by tumor-targeted nanoparticles combined with sunitinib: a new multimodal gene therapy approach for  glioblastoma. J Control Release. 2015;207:31–9.
12. Landen CN Jr, Chavez-Reyes A, Bucana C, et al. Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery. Cancer Res. 2005;65(15):6910–8.
13. Trang P, Wiggins JF, Daige CL, et al. Systemic delivery of tumor suppressor microRNA mimics using a neutral lipid emulsion inhibits lung tumors in mice. Mol Ther. 2011;19(6):1116–22.
14. Wang et al. Nanostructured lipid carriers for MicroRNA delivery in tumor gene therapy. Cancer Cell Int (2018) 18:101.