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IVIS imaging and quantitation of local GFP expression from scaffolds and contained tissue at 24 A. and 72 B. hours; each lane represents samples from a different mouse. Delivered mRNA is all cases was 4 µg. Experimental groups include 40 µm pHEMA PTS control (PTS only), pHEMA PTS with SF:mRNA nanoparticles (PTS SF:mRNA nanoparticles), pHEMA PTS with naked mRNA (PTS-naked mRNA), SF:mRNA nanoparticles subcutaneous bolus injection (SF:mRNA s.c.), and naked mRNA subcutaneous bolus injection (naked mRNA s.c.). (N = 3). C. Average GFP fluorescence signal measures by IVIS; (N = 3) error bars are s.d
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mRNA is increasingly being recognized as a promising alternative to pDNA in gene vaccinations. Only recently, owing to the needs of cancer immunotherapies, has the biomaterials/gene delivery community begun to develop new biomaterial strategies for immunomodulation. Here, we report a novel way to use implantable porous scaffolds as a local gene del...
Citations
... For example, cationic liposomes, such as Lipofectamine MessengerMax (Thermo Fisher Scientific), give variable results, with mRNA transfection efficiencies of ∼0 to 30% in bone marrow-derived myeloid phagocytes (BMMPs). [1][2][3][4][5][6][7] Interestingly, these problems can be largely overcome using lipid nanoparticles (LNPs), 8,9 which have recently emerged as the most clinically advanced nonviral system for nucleic acid delivery. ...
The effective delivery of synthetic RNA into mononuclear phagocytes is a prerequisite for experimental research and therapeutic development. However, traditional methods are highly ineffective and toxic for these cells. Here, we aimed to optimize a transfection protocol for primary bone marrow-derived phagocytes, specifically dendritic cells and macrophages, using lipid nanoparticles generated by microfluidics. Our results show that a lipid mixture similar to that used in Moderna's COVID-19 mRNA vaccine outperforms the others tested. Improved mRNA transfection can be achieved by replacing uridine with methylpseudouridine but not methoxyuridine, which interferes with transfection. The addition of diphenyleneiodonium or apocynin can enhance transfection in a cell type-dependent manner without adverse effects, while apolipoprotein E provides no added value. These optimized transfection conditions can also be used for microRNA agonists and antagonists. In sum, this study offers a straightforward, highly efficient, reproducible, and non-toxic protocol to deliver RNA into different primary mononuclear phagocytes in culture.
... 163 Furthermore, polymers are capable of building scaffolding for mRNA vaccination as well. Studies have proved that scaffold-based mRNA delivery stimulates antigen-specific antibody in mice 164,165 (Figure 7). ...
Messenger ribonucleic acid (mRNA) vaccines are a relatively new class of vaccines that have shown great promise in the immunotherapy of a wide variety of infectious diseases and cancer. In the past two years, SARS-CoV-2 mRNA vaccines have contributed tremendously against SARS-CoV2, which has prompted the arrival of the mRNA vaccine research boom, especially in the research of cancer vaccines. Compared with conventional cancer vaccines, mRNA vaccines have significant advantages, including efficient production of protective immune responses, relatively low side effects, and lower cost of acquisition. In this review, we elaborated on the development of cancer vaccines and mRNA cancer vaccines, as well as the potential biological mechanisms of mRNA cancer vaccines and the latest progress in various tumor treatments, and discussed the challenges and future directions for the field.
... Chen et al. presented the utilization of implantable porous scaffolds as an mRNA vaccine delivery platform [390]. Porous poly (2hydroxyethyl methacrylate) (pHEMA) scaffold containing singlestranded mRNA-Stemfect TM lipoplexes showed superior efficiency in the prolonged local release of mRNA, mRNA uptake by cells, and GFP transgene expression at the implantation site in vivo when compared to the naked RNA-loaded porous scaffold and systemic bolus injection. ...
Implantable drug delivery systems (IDDS) are an attractive alternative to conventional drug administration routes. Oral and injectable drug administration are the most common routes for drug delivery providing peaks of drug concentrations in blood after administration followed by concentration decay after a few hours. Therefore, constant drug administration is required to keep drug levels within the therapeutic window of the drug. Moreover, oral drug delivery presents alternative challenges due to drug degradation within the gastrointestinal tract or first pass metabolism. IDDS can be used to provide sustained drug delivery for prolonged periods of time. The use of this type of systems is especially interesting for the treatment of chronic conditions where patient adherence to conventional treatments can be challenging. These systems are normally used for systemic drug delivery. However, IDDS can be used for localised administration to maximise the amount of drug delivered within the active site while reducing systemic exposure. This review will cover current applications of IDDS focusing on the materials used to prepare this type of systems and the main therapeutic areas of application.
... Moreover, TAMs are being explored for cancer treatment. Although the release of chemotherapeutic drugs from matrices has been studied [26], recent efforts are focused on the use of TAMs as mRNA vaccines with promising results [27][28][29]. ...
... Regarding mesh size, smaller pores and denser structures slow down mRNA release [31,34]. In addition, pore size may condition the chronic inflammation generated by the scaffold [28], a process that directly influences TAM performance. ...
... Furthermore, there is a risk of immunogenicity and disease transmission [65]. Consequently, the use of synthetic polymers such as poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), or hydroxyethyl methacrylate (HEMA) has also been tested [28,34,66]. One of the main benefits of synthetic materials is that they allow good physical-chemical control of their properties [67]. ...
Over two decades of preclinical and clinical experience have confirmed that gene therapy-activated matrices are potent tools for sustained gene modulation at the implantation area. Matrices activated with messenger RNA (mRNA) are the latest development in the area, and they promise an ideal combination of efficiency and safety. Indeed, implanted mRNA-activated matrices allow a sustained delivery of mRNA and the continuous production of therapeutic proteins in situ. In addition, they are particularly interesting to generate proteins acting on intracellular targets, as the translated protein can directly exert its therapeutic function. Still, mRNA-activated matrices are incipient technologies with a limited number of published records, and much is still to be understood before their successful implementation. Indeed, the design parameters of mRNA-activated matrices are crucial for their performance, as they affect mRNA stability, device immunogenicity, translation efficiency, and the duration of the therapy. Critical design factors include matrix composition and its mesh size, mRNA chemical modification and sequence, and the characteristics of the nanocarriers used for mRNA delivery. This review aims to provide some background relevant to these technologies and to summarize both the design space for mRNA-activated matrices and the current knowledge regarding their pharmaceutical performance. Furthermore, we will discuss potential applications of mRNA-activated matrices, mainly focusing on tissue engineering and immunomodulation.
... The first-generation RNA delivery systems used delivery vehicles that appeared in the 1990s such as protamine [29,30], polyethylenimine (PEI), and cationic liposomes [31]. The second generation of delivery systems were mainly the biodegradable, ionizable polymers that emerged in the late 1990s [32,33]. Most of these materials failed to enter clinical research due to their own high toxicity, complex structure, and uncontrollable polymerization. ...
... Nanoparticles with a particle size of less than 20 nm are easily metabolized by the kidney, a large particle size has a greater impact on the transfection effect and safety. As influenced by the particle size, LNPs can only pass through porous endothelial cells, making them the best choice for targeting the liver [31][32][33]. Belliveau et al. achieved reproducible production of LNP-siRNA systems with a 20 nm diameter or larger with a polydispersity index as low as 0.02 using microfluidic mixing at the nanoliter scale with millisecond mixing [34]. LNPs with diameters of 70 nm or larger can also be prepared via microfluidic mixing techniques that use a macroscopic mixing process. ...
Messenger RNA (mRNA), which is composed of ribonucleotides that carry genetic information and direct protein synthesis, is transcribed from a strand of DNA as a template. On this basis, mRNA technology can take advantage of the body’s own translation system to express proteins with multiple functions for the treatment of various diseases. Due to the advancement of mRNA synthesis and purification, modification and sequence optimization technologies, and the emerging lipid nanomaterials and other delivery systems, mRNA therapeutic regimens are becoming clinically feasible and exhibit significant reliability in mRNA stability, translation efficiency, and controlled immunogenicity. Lipid nanoparticles (LNPs), currently the leading non-viral delivery vehicles, have made many exciting advances in clinical translation as part of the COVID-19 vaccines and therefore have the potential to accelerate the clinical translation of gene drugs. Additionally, due to their small size, biocompatibility, and biodegradability, LNPs can effectively deliver nucleic acids into cells, which is particularly important for the current mRNA regimens. Therefore, the cutting-edge LNP@mRNA regimens hold great promise for cancer vaccines, infectious disease prevention, protein replacement therapy, gene editing, and rare disease treatment. To shed more lights on LNP@mRNA, this paper mainly discusses the rational of choosing LNPs as the non-viral vectors to deliver mRNA, the general rules for mRNA optimization and LNP preparation, and the various parameters affecting the delivery efficiency of LNP@mRNA, and finally summarizes the current research status as well as the current challenges. The latest research progress of LNPs in the treatment of other diseases such as oncological, cardiovascular, and infectious diseases is also given. Finally, the future applications and perspectives for LNP@mRNA are generally introduced.
... Another interesting instance was reported by Chen et al. 186 They evaluated different types of polymer-and lipid-based gene carriers including two cationic polymers (in vivo-jetPEI and poly(β-amino ester) (PBAE)) and two lipids (Lip-ofectamineTM-LF-MM and StemfectTM-SF) to improve mRNA vaccine immunization. They also optimized the implantable scaffold systems as mRNA localized delivery. ...
Biodegradable polymers are largely employed in the biomedical field, ranging from tissue regeneration to drug/vaccine delivery. The biodegradable polymers are highly biocompatible and possess negligible toxicity. In addition, biomaterial-based vaccines possess adjuvant properties, thereby enhancing immune responses. This Review introduces the use of different biodegradable polymers and their degradation mechanism. Different kinds of vaccines, as well as the interaction between the carriers with the immune system, then are highlighted. Natural and synthetic biodegradable micro-/nanoplatforms, hydrogels, and scaffolds for local or targeted and controlled vaccine release are subsequently discussed.
... Therefore, we sought to assess the long-term stability of lyophilized collagen scaffolds loaded with mRNA NPs at different temperatures. LMM-formulated mRNA transfections have previously been shown to be compatible with lyophilization [50]. Moreover, since the long-term stability of LNP-formulated mRNA is well-established [51][52][53], we included LMM mRNA NPs as a control for LNP storage stability. ...
... scaffolds loaded with mRNA NPs at different temperatures. LMM-formulated mRNA transfections have previously been shown to be compatible with lyophilization [50]. Moreover, since the long-term stability of LNP-formulated mRNA is well-established [51][52][53], we included LMM mRNA NPs as a control for LNP storage stability. ...
In our aging society, the number of patients suffering from poorly healing bone defects increases. Bone morphogenetic proteins (BMPs) are used in the clinic to promote bone regeneration. However, poor control of BMP delivery and thus activity necessitates high doses, resulting in adverse effects and increased costs. It has been demonstrated that messenger RNA (mRNA) provides a superior alternative to protein delivery due to local uptake and prolonged expression restricted to the site of action. Here, we present the development of porous collagen scaffolds incorporating peptide-mRNA nanoparticles (NPs). Nanoparticles were generated by simply mixing aqueous solutions of the cationic cell-penetrating peptide PepFect14 (PF14) and mRNA. Peptide-mRNA complexes were uniformly distributed throughout the scaffolds, and matrices fully preserved cell attachment and viability. There was a clear dependence of protein expression on the incorporated amount of mRNA. Importantly, after lyophilization, the mRNA formulation in the collagen scaffolds retained activity also at 4 °C over two weeks. Overall, our results demonstrate that collagen scaffolds incorporating peptide-mRNA complexes hold promise as off-the-shelf functional biomaterials for applications in regenerative medicine and constitute a viable alternative to lipid-based mRNA formulations.
... Scaffolds based on various biocompatible and bioresorbable materials with specified physicochemical and biological properties are usually applied to support localized and sustained gene delivery [15]. They provide a microenvironment for infiltration, adhesion, and proliferation of certain cell types, at the same time allowing the embedded Gels 2022, 8,421 2 of 17 genetic constructs to direct cellular differentiation and induce regeneration of damaged tissues [16]. However, despite many studies and real achievements in this field, the search and selection of optimal materials, as well as fabrication methods, to form highly effective gene-activated scaffolds (GAS) are still relevant and challenging in biomaterial science and tissue engineering [17]. ...
Gene therapy is one of the most promising approaches in regenerative medicine to restore damaged tissues of various types. However, the ability to control the dose of bioactive molecules in the injection site can be challenging. The combination of genetic constructs, bioresorbable material, and the 3D printing technique can help to overcome these difficulties and not only serve as a microenvironment for cell infiltration but also provide localized gene release in a more sustainable way to induce effective cell differentiation. Herein, the cell transfection with plasmid DNA directly incorporated into sodium alginate prior to 3D printing was investigated both in vitro and in vivo. The 3D cryoprinting ensures pDNA structure integrity and safety. 3D printed gene-activated scaffolds (GAS) mediated HEK293 transfection in vitro and effective synthesis of model EGFP protein in vivo, thereby allowing the implementation of the developed GAS in future tissue engineering applications.
... In addition, nonviral vectors prevent nonspecific interactions with proteins or non-target cells and promote efficient transport to multiple tissues through targeted delivery [28][29][30]. More recently, numerous non-viral vectors, including polymer [31][32][33], hybrid [34][35][36], and even scaffoldmediated [37] nanoparticles (NPs), have been used and fostered for mRNA delivery. Recent studies have demonstrated that lipid-based NPs as the most frequently used carrier for mRNA delivery, are promising to treat cancer and disease disorders [11,14,[38][39][40]. ...
In the last decade, the development of messenger RNA (mRNA) therapeutics by lipid nanoparticles (LNP) leads to facilitate clinical trial recruitment, which improves the efficacy of treatment modality to a large extent. Although mRNA-LNP vaccine platforms for the COVID-19 pandemic demonstrated high efficiency, safety and adverse effects challenges due to the uncontrolled immune responses and inappropriate pharmacological interventions could limit this tremendous efficacy. The current study reveals the interplay of immune responses with LNP compositions and characterization and clarifies the interaction of mRNA-LNP therapeutics with dendritic, macrophages, neutrophile cells, and complement. Then, pharmacological profiles for mRNA-LNP delivery, including pharmacokinetics and cellular trafficking, were discussed in detail in cancer types and infectious diseases. This review study opens a new and vital landscape to improve multidisciplinary therapeutics on mRNA-LNP through modulation of immunopharmacological responses in clinical trials.
Graphical Abstract
... The liposomes also protected mRNA from degradation and improved its in vivo stability as scaffolds containing SF:mRNA NPs had 2.5-6.7-fold higher retained mRNA than scaffolds containing naked mRNA [106] . Another 3D porous polymer scaffold was developed for the delivery of mRNA vaccine encoding the model ovalbumin antigen. ...
mRNA vaccines have emerged as promising alternative platforms to conventional vaccines. Their ease of production, low cost, safety profile and high potency render them ideal candidates for prevention and treatment of infectious diseases, especially in the midst of pandemics. The challenges that face in vitro transcribed RNA were partially amended by addition of tethered adjuvants or co-delivery of naked mRNA with an adjuvant-tethered RNA. However, it wasn't until recently that the progress made in nanotechnology helped enhance mRNA stability and delivery by entrapment in novel delivery systems of which, lipid nanoparticles. The continuous advancement in the fields of nanotechnology and tissue engineering provided novel carriers for mRNA vaccines such as polymeric nanoparticles and scaffolds. Various studies have shown the advantages of adopting mRNA vaccines for viral diseases and cancer in animal and human studies. Self-amplifying mRNA is considered today the next generation of mRNA vaccines and current studies reveal promising outcomes. This review provides a comprehensive overview of mRNA vaccines used in past and present studies, and discusses future directions and challenges in advancing this vaccine platform to widespread clinical use.
