Synthetic poly(ester amine) and poly(amido amine) nanoparticles for efficient DNA and siRNA delivery to human endothelial cells

Biodegradable poly(ester amine) (PEA)-based and poly(amido amine) (PAA)-based nanoparticles were developed for efficient in vitro siRNA delivery to human umbilical vein endothelial cells (HUVECs). They were screened, characterized, and compared with traditionally studied DNA-containing particles. Several of the polymeric nanoparticles tested were found to be effective for delivering functional siRNA to green fluorescent protein (GFP) + HUVECs, achieving 60%–75% GFP knockdown while maintaining high viability. While PEAs have been used previously to form polyplexes or nanoparticles for DNA delivery, highly effective siRNA delivery in hard-to-transfect human cell types has not been previously reported. PEAs and linear nondendrimeric PAAs were also found to be effective for DNA delivery to HUVECs using GFP-encoding plasmid DNA (up to 50%–60% transfection efficiency). PEAs and PAAs can be separated into groups that form polymeric nanoparticles effective for siRNA delivery, for DNA delivery, or for both

Controlled, pulsatile release of thermostabilized inactivated polio vaccine from PLGA-based microspheres

Many vaccines, such as the inactivated polio vaccine (IPV), must be administered in several doses for full efficacy. Because patient access is a major challenge for vaccination efforts in developing countries, administering multiple doses per patient is impractical in those areas. Single-administration vaccines would greatly improve efforts to vaccinate populations in Third World countries, and the World Health Organization (WHO) Expanded Program for Immunization describes an ideal vaccine as one that is heat-stable, requires only one shot, and is easy to administer. Although already existing technologies, such as microspheres composed of poly(lactic-co-glycolic acid) (PLGA), are able to encapsulate vaccines and release them over an extended period of time up to several weeks, they are not able to maintain antigen stability over the longer time intervals in vivo. Vaccines such as IPV, however, are known to be unstable at elevated temperature, such as the 37°C environment of the body, as well as in the acidic environment of the degrading PLGA microspheres. Please click Additional Files below to see the full abstract

Thermostabilization of inactivated polio vaccine in PLGA-based microspheres for pulsatile release

AbstractVaccines are a critical clinical tool in preventing illness and death due to infectious diseases and are regularly administered to children and adults across the globe. In order to obtain full protection from many vaccines, an individual needs to receive multiple doses over the course of months. However, vaccine administration in developing countries is limited by the difficulty in consistently delivering a second or third dose, and some vaccines, including the inactivated polio vaccine (IPV), must be injected more than once for efficacy. In addition, IPV does not remain stable over time at elevated temperatures, such as those it would encounter over time in the body if it were to be injected as a single-administration vaccine. In this manuscript, we describe microspheres composed of poly(lactic-co-glycolic acid) (PLGA) that can encapsulate IPV along with stabilizing excipients and release immunogenic IPV over the course of several weeks. Additionally, pH-sensitive, cationic dopants such as Eudragit E polymer caused clinically relevant amounts of stable IPV release upon degradation of the PLGA matrix. Specifically, IPV was released in two separate bursts, mimicking the delivery of two boluses approximately one month apart. In one of our top formulations, 1.4, 1.1, and 1.2 doses of the IPV serotype 1, 2, and 3, respectively, were released within the first few days from 50mg of particles. During the delayed, second burst, 0.5, 0.8, and 0.6 doses of each serotype, respectively, were released; thus, 50mg of these particles released approximately two clinical doses spaced a month apart. Immunization of rats with the leading microsphere formulation showed more robust and long-lasting humoral immune response compared to a single bolus injection and was statistically non-inferior from two bolus injections spaced 1 month apart. By minimizing the number of administrations of a vaccine, such as IPV, this technology can serve as a tool to aid in the eradication of polio and other infectious diseases for the improvement of global health

Novel pulsatile-release microparticles for single-injection vaccination

Many controlled release devices are designed to achieve near zero-order release kinetics, however for some applications, such as vaccination, non-continuous or pulsatile release is desired. Such pulsatile release systems may enable the creation of single-injection vaccines that eliminate the need for subsequent booster immunizations by spontaneously releasing antigen at time points that correspond to normal vaccination regimens. This would be especially important in the developing world where a lack of consistent access to healthcare contributes to approximately 1.5 million vaccine-preventable deaths each year.1 Here we present the fabrication and characterization of biodegradable core-shell microparticles that exhibit pulsatile release kinetics due to their unique structure. These particles are produced using a novel fabrication process that combines soft lithography, picoliter dispensing, optical alignment, and a gentle heat-based sintering step to generate microparticles with a biodegradable polymeric shell surrounding an antigen-filled core. By altering the composition (e.g. copolymer ratio or molecular weight) of the poly(lactic-co-glycolic acid) shell, particles can be tuned to release discrete pulses of a model antigen at times ranging from four days to two months. This fabrication method is also compatible with sensitive biologics, such as the inactivated polio virus, which retains \u3e80% of its antigenicity after encapsulation. Further, because the shell of the particle is physically separated from the core, these particles can be filled with any aqueous vaccine solution without affecting release kinetics and be easily scaled via massively parallel fabrication. As a result, these particles have exciting potential as single-injection vaccines that fully mimic the antigen presentation profile of traditional bolus injections administered over the course of months or years. Please click Additional Files below to see the full abstract

Photocrosslinked Bioreducible Polymeric Nanoparticles for Enhanced Systemic siRNA Delivery as Cancer Therapy

Clinical translation of polymer‐based nanocarriers for systemic delivery of RNA has been limited due to poor colloidal stability in the blood stream and intracellular delivery of the RNA to the cytosol. To address these limitations, this study reports a new strategy incorporating photocrosslinking of bioreducible nanoparticles for improved stability extracellularly and rapid release of RNA intracellularly. In this design, the polymeric nanocarriers contain ester bonds for hydrolytic degradation and disulfide bonds for environmentally triggered small interfering RNA (siRNA) release in the cytosol. These photocrosslinked bioreducible nanoparticles (XbNPs) have a shielded surface charge, reduced adsorption of serum proteins, and enable superior siRNA‐mediated knockdown in both glioma and melanoma cells in high‐serum conditions compared to non‐crosslinked formulations. Mechanistically, XbNPs promote cellular uptake and the presence of secondary and tertiary amines enables efficient endosomal escape. Following systemic administration, XbNPs facilitate targeting of cancer cells and tissue‐mediated siRNA delivery beyond the liver, unlike conventional nanoparticle‐based delivery. These attributes of XbNPs facilitate robust siRNA‐mediated knockdown in vivo in melanoma tumors colonized in the lungs following systemic administration. Thus, biodegradable polymeric nanoparticles, via photocrosslinking, demonstrate extended colloidal stability and efficient delivery of RNA therapeutics under physiological conditions, and thereby potentially advance systemic delivery technologies for nucleic acid‐based therapeutics

(3-Aminopropyl)-4-methylpiperazine End-capped Poly(1,4-butanediol diacrylate-co-4-amino-1-butanol)-based Multilayer Films for Gene Delivery

Biodegradable polyelectrolyte surfaces for gene delivery were created through electrospinning of biodegradable polycations combined with iterative solution-based multilayer coating. Poly­(β-amino ester) (PBAE) poly­(1,4-butanediol diacrylate-co-4-amino-1-butanol) end-capped with 1-(3-aminopropyl)-4-methylpiperazine was utilized because of its ability to electrostatically interact with anionic molecules like DNA, its biodegradability, and its low cytotoxicity. A new DNA release system was developed for sustained release of DNA over 24 h, accompanied by high exogenous gene expression in primary human glioblastoma (GB) cells. Electrospinning a different PBAE, poly­(1,4-butanediol diacrylate-co-4,4′-trimethylenedipiperidine), and its combination with polyelectrolyte 1-(3-aminopropyl)-4-methylpiperazine end-capped poly­(1,4-butanediol diacrylate-co-4-amino-1-butanol)-based multilayers are promising for DNA release and intracellular delivery from a surface

T Cell Targeting Biomimetic Polymeric Nanoparticles for mRNA Delivery and Stimulation

T cell immunotherapies have demonstrated robust clinical success in treating some cancers but are not without their challenges. Engineering T cells, such as chimeric antigen receptor (CAR) strategies, has been shown to be a powerful approach to direct the adaptive immune response in cancer. However, these approaches are expensive, time consuming, and inefficient given the need for T cell extraction from the patient, ex vivo engineering, then reinfusion. Genetically modifying T cells in situ would significantly simplify the process and reduce costs. Non-viral polymeric nanoparticles offer a potential vehicle for in situ gene engineering for their well-established efficacy and safety in multiple cell types. Here, we developed a modular, targeted polymeric/lipid NP that can deliver mRNA cargo and effectively stimulate T cells in situ. Using synthetic poly(beta amino ester) (PBAE), mRNA nanoparticles were synthesized by self-assembly in buffer. Particles were subsequently conjugated with either anti-CD3 or both anti-CD3 and anti-CD28, a costimulatory signal, to transfect primary murine T cells isolated from C57BL/6. Our results suggest that both in vitro and in vivo, mRNA-NPs with anti-CD3 conjugated to the surface significantly outperformed unconjugated NPs. In vitro, transfection efficacy of NPs + anti-CD3 reached ~17%, a 3-fold increase over the unconjugated particles. Likewise, particles conjugated with both anti-CD3 and anti-CD28 were able to induce ~5-fold T cell proliferation with minimal toxicity. For in vivo studies, transgenic Ai9 mice were injected systemically with mRNA-NPs encoding a Cre mRNA molecule for tdTomato expression analysis. NPs + anti-CD3 achieved significantly higher transfection in the spleen and lymph nodes than unconjugated particles, and preferentially transfected CD4+ T cells. Unconjugated particles had no significant preference for transfection in CD8+ or CD4+ T cells. Studies are currently underway to further investigate antibody conjugation and apply this T lymphocyte targeted gene delivery platform in situ for immunoengineering applications. </p