Poly(2-propylacrylic acid)/poly(lactic-co-glycolic acid) blend microparticles as a targeted antigen delivery system to direct either CD4+ or CD8+ T cell activation.

Poly(lactic-co-glycolic acid) (PLGA) based microparticles (MPs) are widely investigated for their ability to load a range of molecules with high efficiency, including antigenic proteins, and release them in a controlled manner. Micron-sized PLGA MPs are readily phagocytosed by antigen presenting cells, and localized to endosomes. Due to low pH and digestive enzymes, encapsulated protein cargo is largely degraded and processed in endosomes for MHC-II loading and presentation to CD4+ T cells, with very little antigen delivered into the cytosol, limiting MHC-I antigenic loading and presentation to CD8+ T cells. In this work, PLGA was blended with poly(2-propylacrylic acid) (PPAA), a membrane destabilizing polymer, in order to incorporate an endosomal escape strategy into PLGA MPs as an easily fabricated platform with diverse loading capabilities, as a means to enable antigen presentation to CD8+ T cells. Ovalbumin (OVA)-loaded MPs were fabricated using a water-in-oil double emulsion with a 0% (PLGA only), 3 and 10% PPAA composition. MPs were subsequently determined to have an average diameter of 1 µm, with high loading and a release profile characteristic of PLGA. Bone marrow derived dendritic cells (DCs) were then incubated with MPs in order to evaluate localization, processing, and presentation of ovalbumin. Endosomal escape of OVA was observed only in DC groups treated with PPAA/PLGA blends, which promoted high levels of activation of CD8+ OVA-specific OT-I T cells, compared to DCs treated with OVA-loaded PLGA MPs which were unable activate CD8+ T cells. In contrast, DCs treated with OVA-loaded PLGA MPs promoted OVA-specific OT-II CD4+ T cell activation, whereas PPAA incorporation into the MP blend did not permit CD4+ T cell activation. These studies demonstrate PLGA MP blends containing PPAA are able to provide an endosomal escape strategy for encapsulated protein antigen, enabling the targeted delivery of antigen for tunable presentation and activation of either CD4+ or CD8+ T cells

Development of a targeted and controlled nanoparticle delivery system for FoxO1 inhibitors

Background: Poly (lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) are polymers approved by the United States’ Food and Drug Administration. Drugs for various medical treatments have been encapsulated in PLGA-PEG nanoparticles for targeted delivery and reduction of unwanted side effects. Methods: A flow synthesis method for PLGA-PEG nanoparticles containing FoxO1 inhibitors and adipose vasculature targeting agents was developed. A set of nanoparticles including PLGA and PLGA-PEG-P3 unloaded and drug loaded were generated. The particles were characterized by DLS, fluorescence spectroscopy, TEM, and dialysis. Endotoxin levels were measured using the LAL chromogenic assay. Our approach was compared to over 270 research articles using information extraction tools. Results: Nanoparticle hydrodynamic diameters ranged from 142.4 ±0.4 d.nm to 208.7 ±3.6 d.nm while the polydispersity index was less than 0.500 for all samples (0.057 ±0.021 to 0.369 ±0.038). Zeta potentials were all negative ranging from -4.33 mV to -13.4 mV. Stability testing confirmed that size remained unchanged for up to 4 weeks. For AS1842856, loading was 0.5 mg drug/mL solution and encapsulation efficiency was ~100%. Dialysis indicated burst release of drug in the first 4 hours. Conclusion: PLGA encapsulation of AS1842856 was successful but unsuccessful for the two more hydrophilic drugs. Alternative syntheses such as water/oil/water emulsion or liposomal encapsulation are being considered. Analysis of data from published papers on PLGA nanoparticles indicated that our results were consistent with identified process-structure relationships and few groups reported endotoxin levels even though in vivo testing was performed.https://scholarscompass.vcu.edu/gradposters/1071/thumbnail.jp

Efficient chemotherapy of rat glioblastoma using Doxorubicin-loaded PLGA nanoparticles with different stabilizers

Background: Chemotherapy of glioblastoma is largely ineffective as the blood-brain barrier (BBB) prevents entry of most anticancer agents into the brain. For an efficient treatment of glioblastomas it is necessary to deliver anti-cancer drugs across the intact BBB. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles coated with poloxamer 188 hold great promise as drug carriers for brain delivery after their intravenous injection. In the present study the anti-tumour efficacy of the surfactant-coated doxorubicin-loaded PLGA nanoparticles against rat glioblastoma 101/8 was investigated using histological and immunohistochemical methods. Methodology: The particles were prepared by a high-pressure solvent evaporation technique using 1% polyvinylalcohol (PLGA/PVA) or human serum albumin (PLGA/HSA) as stabilizers. Additionally, lecithin-containing PLGA/HSA particles (Dox-Lecithin-PLGA/HSA) were prepared. For evaluation of the antitumour efficacy the glioblastoma-bearing rats were treated intravenously with the doxorubicin-loaded nanoparticles coated with poloxamer 188 using the following treatment regimen: 3×2.5 mg/kg on day 2, 5 and 8 after tumour implantation; doxorubicin and poloxamer 188 solutions were used as controls. On day 18, the rats were sacrificed and the antitumour effect was determined by measurement of tumour size, necrotic areas, proliferation index, and expression of GFAP and VEGF as well as Isolectin B4, a marker for the vessel density. Conclusion: The results reveal a considerable anti-tumour effect of the doxorubicin-loaded nanoparticles. The overall best results were observed for Dox-Lecithin-PLGA/HSA. These data demonstrate that the poloxamer 188-coated PLGA nanoparticles enable delivery of doxorubicin across the blood-brain barrier in the therapeutically effective concentrations

The effects of bioactive akermanite on physiochemical, drug-delivery, and biological properties of poly(lactide-co-glycolide) beads

Poly(lactide-co-glycolide) (PLGA) beads have been widely studied as a potential drug/protein carrier. The main shortcomings of PLGA beads are that they lack bioactivity and controllable drug-delivery ability, and their acidic degradation by-products can lead to pH decrease in the vicinity of the implants. Akermanite (AK) (Ca(2) MgSi(2) O(7) ) is a novel bioactive ceramic which has shown excellent bioactivity and degradation in vivo. This study aimed to incorporate AK to PLGA beads to improve the physiochemical, drug-delivery, and biological properties of PLGA beads. The microstructure of beads was characterized by SEM. The effect of AK incorporating into PLGA beads on the mechanical strength, apatite-formation ability, the loading and release of BSA, and the proliferation, and differentiation of bone marrow stromal cells (BMSCs) was investigated. The results showed that the incorporation of AK into PLGA beads altered the anisotropic microporous structure into homogenous one and improved their compressive strength and apatite-formation ability in simulated body fluids (SBF). AK neutralized the acidic products from PLGA beads, leading to stable pH value of 7.4 in biological environment. AK led to a sustainable and controllable release of bovine serum albumin (BSA) in PLGA beads. The incorporation of AK into PLGA beads enhanced the proliferation and alkaline phosphatase activity of BMSCs. This study implies that the incorporation of AK into PLGA beads is a promising method to enhance their physiochemical and biological property. AK/PLGA composite beads are a potential bioactive drug-delivery system for bone tissue repair

Targeted protein delivery: carbodiimide crosslinking influences protein release from microparticles incorporated within collagen scaffolds

open access articleTissue engineering response may be tailored via controlled, sustained release of active agents from protein-loaded degradable microparticles incorporated directly within three-dimensional (3D) ice-templated collagen scaffolds. However, the effects of covalent crosslinking during scaffold preparation on the availability and release of protein from the incorporated microparticles have not been explored. Here, we load 3D ice-templated collagen scaffolds with controlled additions of poly-(DL-lactide-co-glycolide) microparticles. We probe the effects of subsequent N-(3-dimethylaminopropyl)- N0-ethylcarbodiimide hydrochloride crosslinking on protein release, using microparticles with different internal protein distributions. Fluorescein isothiocyanate labelled bovine serum albumin is used as a model protein drug. The scaffolds display a homogeneous microparticle distribution, and a reduction in pore size and percolation diameter with increased microparticle addition, although these values did not fall below those reported as necessary for cell invasion. The protein distribution within the microparticles, near the surface or more deeply located within the microparticles, was important in determining the release profile and effect of crosslinking, as the surface was affected by the carbodiimide crosslinking reaction applied to the scaffold. Crosslinking of microparticles with a high proportion of protein at the surface caused both a reduction and delay in protein release. Protein located within the bulk of the microparticles, was protected from the crosslinking reaction and no delay in the overall release profile was seen

Encapsulation of human serum albumin in submicrometer magnetic poly(lactide-co-glycolide) particles as a model system for targeted drug delivery

Two types of iron oxide nanoparticles were synthesized by coprecipitation of Fe(II) and Fe(III) chlorides: water-dispersible γ-Fe2O3 and organic solvent-dispersible oleic acid-coated Fe3O4 particles. The nanoparticles, together with human serum albumin (HSA) serving as a model for a protein-type drug, were then incorporated in poly(lactide-co-glycolide) (PLGA) particles using double emulsion solvent evaporation technique. Morphology, size and particle size distribution of the resulting particles was analyzed by electron microscopy and dynamic light scattering. Iron oxide and HSA encapsulating efficiency was determined by Prussian Blue staining and micro-BCA assay, respectively

Pharmaceutically modified subtilisins withstand acidic conditions and effectively degrade gluten in vivo

Detoxification of gluten immunogenic epitopes is a promising strategy for the treatment of celiac disease. Our previous studies have shown that these epitopes can be degraded in vitro by subtilisin enzymes derived from Rothia mucilaginosa, a natural microbial colonizer of the oral cavity. The challenge is that the enzyme is not optimally active under acidic conditions as encountered in the stomach. We therefore aimed to protect and maintain subtilisin-A enzyme activity by exploring two pharmaceutical modification techniques: PEGylation and Polylactic glycolic acid (PLGA) microencapsulation. PEGylation of subtilisin-A (Sub-A) was performed by attaching methoxypolyethylene glycol (mPEG, 5 kDa). The PEGylation protected subtilisin-A from autolysis at neutral pH. The PEGylated Sub-A (Sub-A-mPEG) was further encapsulated by PLGA. The microencapsulated Sub-A-mPEG-PLGA showed significantly increased protection against acid exposure in vitro. In vivo, gluten immunogenic epitopes were decreased by 60% in the stomach of mice fed with chow containing Sub-A-mPEG-PLGA (0.2mg Sub-A/ g chow) (n=9) compared to 31.9 % in mice fed with chow containing unmodified Sub-A (n=9). These results show that the developed pharmaceutical modification can protect Sub-A from auto-digestion as well as from acid inactivation, thus rendering the enzyme more effective for applications in vivo.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522598/Published versio

A perfusion culture system for assessing bone marrow stromal cell differentiation on PLGA scaffolds for bone repair

Biomaterials development for bone repair is currently hindered by the lack of physiologically relevant in vitro testing systems. Here we describe the novel use of a bi-directional perfusion bioreactor to support the long term culture of human bone marrow stromal cells (BMSCs) differentiated on polylactic co-glycolic acid (PLGA). Primary human BMSCs were seeded onto porous PLGA scaffolds and cultured in static vs. perfusion culture conditions for 21 days in osteogenic vs. control media. PLGA scaffolds were osteoconductive, supporting a mature osteogenic phenotype as shown by the upregulation of Runx2 and the early osteocyte marker E11. Perfusion culture enhanced the expression of osteogenic genes Osteocalcin and Osteopontin. Extracellular matrix deposition and mineralisation were spatially regulated within PLGA scaffolds in a donor dependant manner. This, together with the observed upregulation of Collagen type X suggested an environment permissive for the study of differentiation pathways associated with both intramembranous and endochondral ossification routes of bone healing. This culture system offers a platform to assess BMSC behavior on candidate biomaterials under physiologically relevant conditions. Use of this system may improve our understanding of the environmental cues orchestrating BMSC differentiation and enable fine tuning of biomaterial design as we develop tissue-engineered strategies for bone regeneration

Electrospray synthesis of PLGA TIPS microspheres

We successfully demonstrate the synthesis of polymer microspheres using a single electrospray source, and show their physical characterisation. Electrospray has proven to be a versatile method to manufacture particles, giving tight control over size with quasi-monodisperse size distributions. It is a liquid atomisation technique that generates a monodisperse population of highly charged liquid droplets over a broad size range (nanometres to tens of microns). The droplets contain liquid precursors for the in-flight synthesis of particles, and control over the trajectory of these droplets can be precisely manipulated with the use of electric fields to drive them to a grounded substrate. This study reports a method to synthesize poly(lactic-co-glycolic) acid (PLGA) microspheres using the electrospray and thermally induced phase separation (TIPS) techniques, followed by subsequent freeze-drying, for particle production. These microspheres are of interest as vehicles for controlled drug release systems