Journal of Biotechnology & Biomaterials
Open Access

RNA-based therapies; RNA delivery; Functionalized biomaterials; Lipid nanoparticles; Polymeric carriers; RNA stability; Targeted delivery; Immunogenicity; Therapeutic RNA; Drug delivery systems.

Introduction

RNA-based therapies have emerged as a revolutionary approach in modern medicine, offering the potential to treat a wide range of diseases, from genetic disorders to viral infections and cancer. The fundamental idea behind RNA-based therapies is to use RNA molecules such as mRNA, small interfering RNA (siRNA), and RNA aptamers to modulate cellular processes and correct underlying disease mechanisms. Among these, mRNA therapies have gained significant attention, particularly after the success of COVID-19 vaccines, while siRNA therapies hold promise for silencing disease-causing genes. Despite their therapeutic potential, one of the major challenges in advancing RNA-based therapies to the clinic lies in the efficient and safe delivery of RNA molecules to target cells [1].

RNA is inherently unstable and susceptible to degradation by ribonucleases, and its size and charge make it difficult to cross biological barriers such as the cell membrane. Additionally, once inside the body, RNA molecules must be delivered to specific tissues or cells to exert their therapeutic effects without causing off-target effects or triggering an immune response. The traditional approach of using viral vectors for RNA delivery, though effective, raises concerns regarding immunogenicity, toxicity, and manufacturing complexity. These limitations have driven researchers to explore non-viral delivery systems that can provide greater control over the delivery process, improve targeting specificity, and reduce adverse reactions.

Functionalized biomaterials have emerged as a promising solution to overcome these challenges. These materials, which include lipids, polymers, and hydrogels, can be engineered to encapsulate RNA molecules and protect them from degradation. Functionalization, which involves modifying the surface of biomaterials with specific ligands, allows for targeting particular cell types or tissues, enhancing delivery efficiency. Lipid nanoparticles (LNPs), for instance, have been widely used for mRNA delivery, especially in the context of COVID-19 vaccines, due to their ability to efficiently deliver RNA while minimizing immune activation. Polymeric carriers, such as polyethylenimine (PEI) and dendrimers, offer advantages in terms of scalability and customization, providing flexibility for various RNA-based applications  [ 2, 3].

Despite the progress made with functionalized biomaterials, several challenges remain. The efficiency of RNA delivery to specific cells and tissues still requires significant optimization, particularly for systemic delivery. Immune responses to foreign biomaterials or RNA molecules themselves remain a concern, with potential for inflammation and other adverse effects. Furthermore, large-scale manufacturing of RNA formulations and functionalized carriers that meet stringent regulatory standards continues to be a significant hurdle. Researchers are actively exploring strategies to enhance RNA stability, minimize toxicity, and improve targeting precision. Additionally, the development of smart biomaterials that can respond to environmental cues, such as pH or temperature changes, is an exciting area of investigation for achieving more controlled and efficient RNA delivery.

This review provides an overview of the current landscape of functionalized biomaterials used for RNA delivery, discussing their advantages, limitations, and ongoing advancements. We aim to highlight the multifaceted approach required to address the complexities of RNA delivery, with a focus on overcoming challenges related to stability, cellular uptake, targeting specificity, and immunogenicity. By examining the current state of RNA-based therapies and their delivery systems, we will also explore future opportunities for improving therapeutic outcomes and broadening the applications of RNA therapies across a range of diseases.

Materials and Methods

This section outlines the materials and methods utilized in exploring the use of functionalized biomaterials for the delivery of RNA-based therapies. The methodologies presented here encompass the synthesis, characterization, and evaluation of different biomaterials, as well as their interactions with RNA molecules. The effectiveness of RNA delivery systems is assessed through a combination of in vitro and in vivo studies, including cell culture experiments, RNA stability assessments, cellular uptake analysis, and therapeutic efficacy evaluation  [ 4].

Synthesis of functionalized biomaterials

Lipid Nanoparticles (LNPs): LNPs are synthesized using a modified solvent evaporation method. The lipids (such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and cholesterol) are dissolved in an organic solvent (ethanol), and the RNA payload (such as mRNA or siRNA) is added to the lipid solution. The mixture is then rapidly injected into an aqueous phase containing poly(ethylene glycol) (PEG) to form LNPs through a process of solvent evaporation. Functionalization is achieved by incorporating targeting ligands (e.g., peptides, antibodies, or aptamers) into the lipid bilayer or PEG chain to direct the particles to specific cell types.

Polymeric Carriers: Polymeric carriers such as polyethylenimine (PEI) or dendrimers are synthesized through the polymerization of monomers. PEI is synthesized by reacting ethylenimine with a crosslinking agent. For functionalization, targeting groups (e.g., folic acid, peptides) are conjugated to the polymer using covalent linkages. Dendrimers are synthesized through iterative steps of branching, and functional groups are attached to the terminal ends of the dendritic structure for RNA encapsulation and cell-specific targeting.

Hydrogels: Hydrogels are prepared by crosslinking natural or synthetic polymers, such as alginate, polyethylene glycol (PEG), or chitosan, in the presence of a crosslinking agent (e.g., calcium chloride for alginate). Functionalization is achieved by modifying the polymer with bioactive molecules such as peptides, antibodies, or aptamers. RNA is loaded into the hydrogel matrix by mixing the RNA solution with the polymer solution prior to gelation

RNA preparation and loading

RNA Isolation and Purification: RNA molecules (e.g., mRNA, siRNA) are synthesized or obtained from commercial sources. mRNA is typically synthesized using an in vitro transcription kit, while siRNA is chemically synthesized. The RNA is purified using a silica-based column or ethanol precipitation methods to remove impurities and ensure high-quality RNA suitable for delivery.

Encapsulation of RNA into Biomaterials: RNA molecules are loaded into functionalized biomaterials through electrostatic assembly, coacervation, or solvent evaporation methods. For LNPs, RNA is typically mixed with the lipid phase during the nanoparticle formation process. In the case of polymeric carriers, RNA is mixed with the polymeric solution, forming complexes by electrostatic interaction or ionic gelation. Hydrogels are loaded with RNA by dispersing RNA molecules within the polymer matrix during the gelation process [5].

Characterization of biomaterials

Particle Size and Zeta Potential: The size distribution and surface charge (zeta potential) of the functionalized biomaterials are measured using dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively. These measurements provide insights into the stability, colloidal properties, and the ability of the particles to interact with cell membranes.

RNA Encapsulation Efficiency: The RNA encapsulation efficiency is determined by isolating free RNA from the biomaterial formulation through dialysis or centrifugation, followed by quantifying the amount of encapsulated RNA using spectrophotometry (e.g., UV-Vis at 260 nm). The encapsulation efficiency is calculated as the ratio of the RNA incorporated into the biomaterials to the total RNA used in the formulation [6].

Surface Morphology: The surface morphology of the functionalized biomaterials is evaluated using scanning electron microscopy (SEM) or transmission electron microscopy (TEM). These imaging techniques provide detailed information on the particle size, shape, and surface characteristics.

Stability and Release Kinetics: RNA-loaded biomaterials are incubated under physiological conditions (pH 7.4, 37°C) for various time points, and RNA release is measured using spectrophotometry or gel electrophoresis. The stability of the RNA in the biomaterials is assessed by examining the integrity of the RNA before and after storage, as well as during the release process.

In vitro studies

Cell Culture and Cytotoxicity Assays: Human cell lines (e.g., HeLa, HepG2, or primary cells) are cultured in appropriate growth media with supplements at 37°C and 5% CO2. Cytotoxicity of RNA-loaded biomaterials is evaluated using MTT or WST-1 assays, which assess cell viability after exposure to the nanoparticles or hydrogels. Control groups include untreated cells and cells treated with free RNA or bare biomaterials.

Cellular Uptake and Internalization: Cellular uptake is evaluated using fluorescence microscopy or flow cytometry. For fluorescence microscopy, biomaterials are labeled with a fluorescent dye (e.g., rhodamine or Cy5), and the uptake is visualized under a fluorescence microscope. Flow cytometry is used to quantify the uptake efficiency by measuring the fluorescence intensity of cells treated with fluorescently labeled biomaterials  [ 7].

Gene Silencing and Expression Analysis (for siRNA delivery): For siRNA-based therapies, gene silencing is assessed by measuring the expression of the target gene. RNA is extracted from treated cells, and quantitative PCR (qPCR) or Western blot analysis is performed to measure the levels of the target mRNA or protein, respectively.

In vivo studies

Animal Models: In vivo experiments are conducted using appropriate animal models (e.g., mice or rats), and ethical approval is obtained for all animal studies. RNA-loaded functionalized biomaterials are administered via systemic or local injection, depending on the delivery system being studied. The animals are monitored for signs of toxicity and clinical symptoms.

Biodistribution and Imaging: To evaluate the biodistribution of RNA-loaded biomaterials, nanoparticles or hydrogels are labeled with a radioactive or fluorescent tracer. Non-invasive imaging techniques, such as bioluminescence, fluorescence imaging, or positron emission tomography (PET), are employed to track the accumulation of the biomaterials in different tissues over time  [ 8, 9].

Therapeutic Efficacy: The therapeutic efficacy of the RNA-based therapy is assessed based on the intended disease model. For example, mRNA-based vaccines are evaluated by measuring immune response markers (e.g., antibody titers or T cell activation), while siRNA therapies are assessed by measuring the reduction in target gene expression or tumor growth inhibition.

Statistical Analysis: Data from in vitro and in vivo studies are analyzed using appropriate statistical methods, such as Student’s t-test or analysis of variance (ANOVA), to determine the significance of differences between experimental groups. Results are expressed as mean ± standard deviation (SD), and statistical significance is set at p < 0.05.

By employing these materials and methods, this study aims to systematically investigate the potential of functionalized biomaterials for the efficient and targeted delivery of RNA-based therapies, addressing key challenges and providing insights into their future clinical applications  [ 10].

Discussion

The development of RNA-based therapies has made significant strides in recent years, particularly with the success of mRNA vaccines against COVID-19. However, the clinical translation of these therapies is hampered by the challenges associated with RNA delivery. Functionalized biomaterials have emerged as a promising solution to address these hurdles, with advances in lipid nanoparticles (LNPs), polymeric carriers, and hydrogels offering new possibilities for efficient, targeted delivery.

Lipid nanoparticles, especially those used in mRNA vaccines, have shown considerable success in encapsulating RNA molecules, protecting them from degradation, and enhancing cellular uptake. The key to their success lies in their ability to self-assemble into nanosized particles that can efficiently merge with cell membranes. Functionalization of LNPs, with targeting ligands such as antibodies or peptides, offers a strategy to improve specificity, directing RNA to specific cell types or tissues. Despite their success, LNPs have limitations, including potential immune responses, stability issues under storage, and challenges in scaling up production for clinical use.

Polymeric carriers, such as polyethylenimine (PEI), dendrimers, and chitosan, provide an alternative to lipid-based systems. These materials offer the advantage of being more easily customizable and scalable, with modifications in structure and surface charge enabling fine-tuning of RNA encapsulation, release profiles, and cellular interactions. However, the cytotoxicity of some polymeric carriers, particularly PEI, remains a concern, as high doses may lead to cell membrane disruption. Ongoing research is focused on improving the biocompatibility and toxicity profile of these carriers, with strategies including the development of biodegradable polymers and the conjugation of biocompatible coatings.

Hydrogels, with their ability to provide sustained and localized release of RNA, are particularly useful for applications that require prolonged delivery, such as gene therapies or cancer treatments. These materials can be engineered to release RNA in response to environmental stimuli, such as pH or temperature changes. The major challenge for hydrogel-based systems is achieving efficient RNA loading without compromising the gel’s mechanical properties, as well as ensuring the stability of RNA molecules over extended periods. Additionally, hydrogel systems can be difficult to scale up and manufacture under cGMP (current Good Manufacturing Practice) conditions, which limits their broad application.

A common challenge across all functionalized biomaterials is the stability of RNA. RNA is highly susceptible to degradation by ribonucleases, which makes it difficult to deliver intact and functional RNA to target cells. Functionalized biomaterials provide a protective environment for RNA, but the integrity of the RNA must be maintained throughout the delivery process. Strategies to improve RNA stability include optimizing the materials used for encapsulation and employing formulations that provide protection from enzymatic degradation.

Another critical challenge is achieving efficient cellular uptake without triggering unwanted immune responses. While functionalized biomaterials can enhance cellular targeting, ensuring that these materials do not activate the immune system or cause toxicity remains a major concern. The development of "stealth" biomaterials, which reduce recognition by immune cells, and the incorporation of targeting ligands that selectively bind to receptors on target cells, are strategies being explored to minimize immunogenicity.

The scalability and reproducibility of RNA delivery systems remain significant barriers to widespread clinical use. The complex nature of RNA molecules, combined with the need for precise control over particle size, charge, and surface properties, makes large-scale manufacturing a challenge. Regulatory approval processes also require thorough testing of the safety and efficacy of these delivery systems, which can be time-consuming and costly. However, with advancements in manufacturing technologies, such as microfluidic devices for nanoparticle synthesis, and increased investment in RNA delivery research, these challenges may become more manageable in the future.

Despite these obstacles, the potential of RNA-based therapies is undeniable. The combination of functionalized biomaterials with RNA delivery systems opens up new possibilities for treating genetic disorders, cancers, and infectious diseases. Furthermore, advances in gene editing technologies, such as CRISPR/Cas9, will likely increase the demand for efficient RNA delivery systems that can precisely target and modify specific genes in vivo. The integration of RNA therapies with functionalized biomaterials will be key to overcoming current limitations and realizing the full therapeutic potential of RNA-based interventions.

In conclusion, functionalized biomaterials offer significant opportunities for improving the delivery of RNA-based therapies. While challenges such as RNA stability, immune responses, and scalability persist, ongoing research is addressing these issues through the development of more efficient and biocompatible materials. With continued innovation in biomaterials and RNA delivery technologies, the future of RNA-based therapies looks promising, with the potential to revolutionize the treatment of a wide range of diseases.

Conclusion

Functionalized biomaterials have become a cornerstone in the development of RNA-based therapies, offering solutions to many of the challenges associated with RNA delivery. The advances in lipid nanoparticles (LNPs), polymeric carriers, and hydrogels have demonstrated the potential to encapsulate, protect, and efficiently deliver RNA molecules to target cells, thus unlocking new possibilities in the treatment of genetic disorders, cancer, and infectious diseases. These materials enable the stabilization of RNA, enhance cellular uptake, and provide controlled release, addressing key obstacles such as RNA degradation, immunogenicity, and cellular targeting.

However, while significant progress has been made, there are still several challenges that need to be addressed. The stability of RNA remains a critical issue, as RNA molecules are highly susceptible to enzymatic degradation. Although functionalized biomaterials can protect RNA during delivery, optimizing these systems to ensure long-term stability and effective release is still an ongoing area of research. Additionally, the potential for immune responses to both RNA molecules and delivery systems requires further investigation, as does the need for minimizing cytotoxicity and ensuring the biocompatibility of these materials, particularly for systemic applications.

Another important challenge lies in scaling up the production of RNA-loaded functionalized biomaterials. Manufacturing these systems at a large scale, while maintaining consistency and quality, is essential for their clinical application. Regulatory hurdles related to safety, efficacy, and manufacturing practices also need to be navigated to bring these therapies to market. With the increasing demand for RNA-based treatments, the development of more cost-effective, reproducible, and scalable delivery systems is essential for widespread adoption.

Despite these challenges, the opportunities presented by functionalized biomaterials are vast. The combination of RNA-based therapies with innovative biomaterials offers a transformative approach to treating diseases that were once considered untreatable. Future research will likely focus on further enhancing the specificity of delivery, improving the targeting of specific tissues and cell types, and reducing off-target effects. In addition, the integration of RNA-based therapies with gene editing technologies, such as CRISPR, will require even more refined delivery systems capable of precisely modulating gene expression.

Ultimately, the success of RNA-based therapies will depend on the continued collaboration between material scientists, biologists, and clinicians to refine the design and application of functionalized biomaterials. As these systems evolve, they hold the promise of revolutionizing the landscape of modern medicine, offering new hope for treating a broad spectrum of diseases with unprecedented precision and efficiency. With continued innovation and research, RNA-based therapies, coupled with functionalized biomaterials, have the potential to be at the forefront of the next wave of medical breakthroughs.

Conflict of interest

None

Acknowledgment

None

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