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

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

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

Rapid, Single-Cell Analysis and Discovery of Vectored mRNA Transfection In Vivo with a loxP-Flanked tdTomato Reporter Mouse

mRNA therapeutics hold promise for the treatment of diseases requiring intracellular protein expression and for use in genome editing systems, but mRNA must transfect the desired tissue and cell type to be efficacious. Nanoparticle vectors that deliver the mRNA are often evaluated using mRNA encoding for reporter genes such as firefly luciferase (FLuc); however, single-cell resolution of mRNA expression cannot generally be achieved with FLuc, and, thus, the transfected cell populations cannot be determined without additional steps or experiments. To more rapidly identify which types of cells an mRNA formulation transfects in vivo, we describe a Cre recombinase (Cre)-based system that permanently expresses fluorescent tdTomato protein in transfected cells of genetically modified mice. Following in vivo application of vectored Cre mRNA, it is possible to visualize successfully transfected cells via Cre-mediated tdTomato expression in bulk tissues and with single-cell resolution. Using this system, we identify previously unknown transfected cell types of an existing mRNA delivery vehicle in vivo and also develop a new mRNA formulation capable of transfecting lung endothelial cells. Importantly, the same formulations with mRNA encoding for fluorescent protein delivered to wild-type mice did not produce sufficient signal for any visualization in vivo, demonstrating the significantly improved sensitivity of our Cre-based system. We believe that the system described here may facilitate the identification and characterization of mRNA delivery vectors to new tissues and cell types

Great expectations: private sector activity in tissue engineering, regenerative medicine, and stem cell therapeutics.

This report draws upon data from a variety of sources to provide a detailed estimate of the current scope of private sector development and commercial activity in the aggregate field comprising tissue engineering, regenerative medicine, and stem cell therapeutics. Economic activity has grown a remarkable fivefold in the past 5 years. As of mid-2007 approximately 50 firms or business units with over 3000 employees offered commercial tissue-regenerative products or services with generally profitable annual sales in excess of $1.3 billion. Well over a million patients have been treated with these products. In addition, 110 development-stage companies with over 55 products in FDA-level clinical trials and other preclinical stages employed approximately 2500 scientists or support personnel and spent 850 million development dollars in 2007. These totals represent a remarkable recovery from the downturn of 2000-2002, at which time tissue engineering was in shambles because of disappointing product launches, failed regulatory trials, and the general investment pullback following the dot-com crash. Commercial success has resulted in large measure from identification of products that are achievable with available technology and under existing regulatory guidelines. Development-stage firms have become much more adept at risk management. The resilience of the field, as well as its current breadth and diversity, augurs well for the future of regenerative medicine

Micromolding of Thermoplastic Polymers for Direct Fabrication of Discrete, Multilayered Microparticles

Soft lithography provides a convenient and effective method for the fabrication of microdevices with uniform size and shape. However, formation of an embossed, connective film as opposed to discrete features has been an enduring shortcoming associated with soft lithography. Removing this residual layer requires additional postprocessing steps that are often incompatible with organic materials. This limits adaptation and widespread realization of soft lithography for broader applications particularly in drug discovery and drug delivery fields. A novel and versatile approach is demonstrated that enables fabrication of discrete, multilayered, fillable, and harvestable microparticles directly from any thermoplastic polymer, even at very high molecular weights. The approach, isolated microparticle replication via surface-segregating polymer blend mold, utilizes a random copolymer additive, designed with a highly fluorinated segment that, when blended with the mold's matrix, spontaneously orients to the surface conferring an extremely low surface energy and nonwetting properties to the template. The extremely nonwetting properties of the mold are further utilized to load soluble biologics directly into the built-in microwells in a rapid and efficient manner using an innovative screen-printing approach. It is believed that this approach holds promise for fabrication of large-array, 3D, complex microstructures, and is a significant step toward clinical translation of microfabrication technologies

Layer-by-Layer Encapsulation of Probiotics for Delivery to the Microbiome

The gastrointestinal (GI) microbiome is widely investigated for its role in many diseases. However, technologies designed for microbiome delivery are lacking. Here, a layer-by-layer (LbL) approach is reported for probiotic encapsulation to protect probiotics against GI tract insults and improve their adhesion and growth on the intestines. These advantages translate to significantly enhanced survival of LbL-probiotics in vivo

mRNA vaccine delivery using lipid nanoparticles

mRNA vaccines elicit a potent immune response including antibodies and cytotoxic T cells. mRNA vaccines are currently evaluated in clinical trials for cancer immunotherapy applications, but also have great potential as prophylactic vaccines. Efficient delivery of mRNA vaccines will be key for their success and translation to the clinic. Among potential nonviral vectors, lipid nanoparticles are particularly promising. Indeed, lipid nanoparticles can be synthesized with relative ease in a scalable manner, protect the mRNA against degradation, facilitate endosomal escape, can be targeted to the desired cell type by surface decoration with ligands, and as needed, can be codelivered with adjuvants.National Institutes of Health (U.S.) (EB 000244