Polymer-Coated Extracellular Vesicles for Selective Codelivery of Chemotherapeutics and siRNA to Cancer Cells.

Combination therapies involving small-interfering RNA (siRNA)-mediated gene silencing and small-molecule drugs are of high interest for cancer treatment. Among the current gene delivery carriers, cell-derived extracellular vesicles (EVs) are particularly promising candidates due to their high biocompatibility, low immunogenicity, in vivo stability, and inherent targeting ability. Here, we developed a multifunctional EV platform capable of selective codelivery of siRNA and doxorubicin (DOX) to cancer cells. siRNA was first loaded into engineered lipid-hybridized EVs (eEVs) to serve as a core. Subsequently, DOX was incorporated into a polyelectrolyte shell surrounding eEVs, which was deposited by layer-by-layer (LbL) assembly. This approach resulted in the production of a stable EV-polymer complex (LbL-eEV) with a diameter of 140.2 ± 9.0 nm and zeta potential of +22.1 ± 0.5 mV. Experiments were performed to assess cellular uptake, cytotoxicity, and gene silencing efficacy in lung adenocarcinoma cells (A549), with noncancerous fibroblast cells (CCL-210) used as a control. The results demonstrated that the LbL-eEV complex can traffic through cells and release siRNA in the cytoplasm, while delivered DOX enters nuclei to induce programmed cell death. Moreover, the inherent selectivity of the particles for cancer cells resulted in effective gene silencing and cancer killing efficiency with reduced cytotoxicity to normal cells. Synchronous delivery of siRNA and DOX was also verified by flow cytometry analysis of single cells. In summary, these data provide a proof of concept for engineering EVs to deliver multiple therapeutics and suggest that LbL-eEVs are a promising drug delivery platform for targeting cancer.

Nanoengineered Light-Activatable Polybubbles for On-Demand Therapeutic Delivery.

Vaccine coverage is severely limited in developing countries due to inefficient protection of vaccine functionality as well as lack of patient compliance to receive the additional booster doses. Thus, there is an urgent need to design a thermostable vaccine delivery platform that also enables release of the bolus after predetermined time. Here, the formation of injectable and light-activatable polybubbles for vaccine delivery is reported. In vitro studies show that polybubbles enable delayed burst release, irrespective of cargo types, namely small molecule and antigen. The extracorporeal activation of polybubbles is achieved by incorporating near-infrared (NIR)-sensitive gold nanorods (AuNRs). Interestingly, light-activatable polybubbles can be used for on-demand burst release of cargo. In vitro, ex vivo, and in vivo studies demonstrate successful activation of AuNR-loaded polybubbles. Overall, the light-activatable polybubble technology can be used for on-demand delivery of various therapeutics including small molecule drugs, immunologically relevant protein, peptide antigens, and nucleic acids.

Engineered extracellular vesicles with synthetic lipids via membrane fusion to establish efficient gene delivery.

The low yield of extracellular vesicle (EV) secretion is a major obstacle for mass production and limits their potential for clinical applications as a drug delivery platform. Here, we mass produced engineered extracellular vesicles (eEVs) by fusing the surface composition of EVs with lipid-based materials via a membrane extrusion technique. A library of lipids (DOTAP, POPC, DPPC and POPG) was fused with EVs to form a hybrid-lipid membrane structure. Uniform lamellar vesicles with a controlled size around 100 nm were obtained in this study. Particle number characterization revealed this extrusion method allowed a 6- to 43-fold increase in numbers of vesicles post- isolation. Further, exogenous siRNA was successfully loaded into engineered vesicles with ~ 15% - 20% encapsulation efficiency using electroporation technique. These engineered extracellular vesicles sustained a 14-fold higher cellular uptake to lung cancer cells (A549) and achieved an effective gene silencing effect comparable to commercial Lipofectamine RNAiMax. Our results demonstrate the surface composition and functionality of EVs can be tuned by extrusion with lipids and suggest the engineered vesicles can be a potential substitute as gene delivery carriers while being able to be mass produced to a greater degree with retained targeting capabilities of EVs.

Engineering DNA vaccines against infectious diseases.

Engineering vaccine-based therapeutics for infectious diseases is highly challenging, as trial formulations are often found to be nonspecific, ineffective, thermally or hydrolytically unstable, and/or toxic. Vaccines have greatly improved the therapeutic landscape for treating infectious diseases and have significantly reduced the threat by therapeutic and preventative approaches. Furthermore, the advent of recombinant technologies has greatly facilitated growth within the vaccine realm by mitigating risks such as virulence reversion despite making the production processes more cumbersome. In addition, seroconversion can also be enhanced by recombinant technology through kinetic and nonkinetic approaches, which are discussed herein. Recombinant technologies have greatly improved both amino acid-based vaccines and DNA-based vaccines. A plateau of interest has been reached between 2001 and 2010 for the scientific community with regard to DNA vaccine endeavors. The decrease in interest may likely be attributed to difficulties in improving immunogenic properties associated with DNA vaccines, although there has been research demonstrating improvement and optimization to this end. Despite improvement, to the extent of our knowledge, there are currently no regulatory body-approved DNA vaccines for human use (four vaccines approved for animal use). This article discusses engineering DNA vaccines against infectious diseases while discussing advantages and disadvantages of each, with an emphasis on applications of these DNA vaccines. STATEMENT OF SIGNIFICANCE: This review paper summarizes the state of the engineered/recombinant DNA vaccine field, with a scope entailing "Engineering DNA vaccines against infectious diseases". We endeavor to emphasize recent advances, recapitulating the current state of the field. In addition to discussing DNA therapeutics that have already been clinically translated, this review also examines current research developments, and the challenges thwarting further progression. Our review covers: recombinant DNA-based subunit vaccines; internalization and processing; enhancing immune protection via adjuvants; manufacturing and engineering DNA; the safety, stability and delivery of DNA vaccines or plasmids; controlling gene expression using plasmid engineering and gene circuits; overcoming immunogenic issues; and commercial successes. We hope that this review will inspire further research in DNA vaccine development.

Laminin modification subretinal bio-scaffold remodels retinal pigment epithelium-driven microenvironment in vitro and in vivo.

Advanced age-related macular degeneration (AMD) may lead to geographic atrophy or fibrovascular scar at macular, dysfunctional retinal microenvironment, and cause profound visual loss. Recent clinical trials have implied the potential application of pluripotent cell-differentiated retinal pigment epithelial cells (dRPEs) and membranous scaffolds implantation in repairing the degenerated retina in AMD. However, the efficacy of implanted membrane in immobilization and supporting the viability and functions of dRPEs, as well as maintaining the retinal microenvironment is still unclear. Herein we generated a biomimetic scaffold mimicking subretinal Bruch's basement from plasma modified polydimethylsiloxane (PDMS) sheet with laminin coating (PDMS-PmL), and investigated its potential functions to provide a subretinal environment for dRPE-monolayer grown on it. Firstly, compared to non-modified PDMS, PDMS-PmL enhanced the attachment, proliferation, polarization, and maturation of dRPEs. Second, PDMS-PmL increased the polarized tight junction, PEDF secretion, melanosome pigment deposit, and phagocytotic-ability of dRPEs. Third, PDMS-PmL was able to carry a dRPEs/photoreceptor-precursors multilayer retina tissue. Finally, the in vivo subretinal implantation of PDMS-PmL in porcine eyes showed well-biocompatibility up to 2-year follow-up. Notably, multifocal ERGs at 2-year follow-up revealed well preservation of macular function in PDMS-PmL, but not PDMS, transplanted porcine eyes. Trophic PEDF secretion of macular retina in PDMS-PmL group was also maintained to preserve retinal microenvironment in PDMS-PmL eyes at 2 year. Taken together, these data indicated that PDMS-PmL is able to sustain the physiological morphology and functions of polarized RPE monolayer, suggesting its potential of rescuing macular degeneration in vivo.

Thermosensitive chitosan-based hydrogel as a topical ocular drug delivery system of latanoprost for glaucoma treatment.

Ocular hypertension is a major risk factor for the development and progression of glaucoma. Frequent and long-term application of latanoprost often causes undesirable local side effects, which are a major cause of therapeutic failure due to loss of persistence in using this glaucoma medical therapy. In the present study, we developed a thermosensitive chitosan-based hydrogel as a topical eye drop formulation for the sustained release of latanoprost to control ocular hypertension. The developed formulation without preservatives may improve compliance and possibly even efficacy. The results of this study support its biocompatibility and sustained-release profile both in vitro and in vivo. After topical application of latanoprost-loaded hydrogel, triamcinolone acetonide-induced elevated intraocular pressure was significantly decreased within 7 days and remained at a normal level for the following 21 days in rabbit eyes. This newly developed chitosan-based hydrogel may provide a non-invasive alternative to traditional anti-glaucoma eye drops for glaucoma treatment.