Medical applications of biopolymer nanofibers.

A wide array of biomedical applications, extending from the fabrication of implant materials to targeted drug delivery, can be attributed to polymers. The utilization of chemical monomers to form polymers, such as polypropylene, polystyrene, and polyethylene, can provide high mechanical stability to them and they can be utilized for diverse electronic or thermal applications. However, certain chemical-based synthetic polymers are toxic to humans, animals, plants, and microbial cells. Thus, biopolymers have been introduced as an alternative to make them utilizable for biomedical applications. Even though biopolymers possess beneficial biomedical applications, they are not stable in biological fluids and exhibit toxicity in certain cases. Recent advances in nanotechnology have expanded its applicational significance in various domains, especially in the evolution of biopolymers to transform them into nanoparticles for numerous biomedical applications. In particular, biopolymers are fabricated as nanofibers to enhance their biological properties and to be utilized for exclusive biomedical applications. The aim of this review is to present an overview of various biopolymer nanofibers and their distinct synthesis approaches. In addition, the medical applications of biopolymer nanofibers, including antimicrobial agents, drug delivery systems, biosensor production, tissue engineering, and implant fabrication, are also discussed.

Plant celluloses, hemicelluloses, lignins, and volatile oils for the synthesis of nanoparticles and nanostructured materials.

A huge variety of plants are harvested worldwide and their different constituents can be converted into a broad range of bionanomaterials. In parallel, much research effort in materials science and engineering is focused on the formation of nanoparticles and nanostructured materials originating from agricultural residues. Cellulose (40-50%), hemicellulose (20-40%), and lignin (20-30%) represent major plant ingredients and many techniques have been described that separate the main plant components for the synthesis of nanocelluloses, nano-hemicelluloses, and nanolignins with divergent and controllable properties. The minor components, such as essential oils, could also be used to produce non-toxic metal and metal oxide nanoparticles with high bioavailability, biocompatibility, and/or bioactivity. This review describes the chemical structure, the physical and chemical properties of plant cell constituents, different techniques for the synthesis of nanocelluloses, nanohemicelluloses, and nanolignins from various lignocellulose sources and agricultural residues, and the extraction of volatile oils from plants as well as their use in metal and metal oxide nanoparticle production and emulsion preparation. Furthermore, details about the formation of activated carbon nanomaterials by thermal treatment of lignocellulose materials, a few examples of mineral extraction from agriculture waste for nanoparticle fabrication, and the emerging applications of plant-based nanomaterials in different fields, such as biotechnology and medicine, environment protection, environmental remediation, or energy production and storage, are also included. This review also briefly discusses the recent developments and challenges of obtaining nanomaterials from plant residues, and the issues surrounding toxicity and regulation.

Versatility of polymethacrylate monoliths for chromatographic purification of biomolecules.

Polymethacrylate monoliths, specifically poly(glycidyl methacrylate-co-ethylene dimethacrylate) or poly(GMA-co-EDMA) monoliths, are a new generation of chromatographic supports and are significantly different from conventional particle-based adsorbents, membranes, and other monolithic supports for biomolecule purification. Similar to other monoliths, polymethacrylate monoliths possess large pores which allow convective flow of mobile phase and result in high flow rates at reduced pressure drop, unlike particulate supports. The simplicity of the adsorbent synthesis, pH resistance, and the ease and flexibility of tailoring their pore size to that of the target biomolecule are the key properties which differentiate polymethacrylate monoliths from other monoliths. Polymethacrylate monoliths are endowed with reactive epoxy groups for easy functionalization (with anion-exchange, hydrophobic, and affinity ligands) and high ligand retention. In this review, the structure and performance of polymethacrylate monoliths for chromatographic purification of biomolecules are evaluated and compared to those of other supports. The development and use of polymethacrylate monoliths for research applications have grown rapidly in recent times and have enabled the achievement of high through-put biomolecule purification on semi-preparative and preparative scales.

Binding Characterization of Aptamer-Drug Layered Microformulations and In Vitro Release Assessment.

Efficient delivery of adequate active ingredients to targeted malignant cells is critical, attributing to recurrent biophysical and biochemical challenges associated with conventional pharmaceutical delivery systems. These challenges include drug leakage, low targeting capability, high systemic cytotoxicity, and poor pharmacokinetics and pharmacodynamics. Targeted delivery system is a promising development to deliver sufficient amounts of drug molecules to target cells in a controlled release pattern mode. Aptameric ligands possess unique affinity targeting capabilities which can be exploited in the design of high pay-load drug formulations to navigate active molecules to the malignant sites. This study focuses on the development of a copolymeric and multifunctional drug-loaded aptamer-conjugated poly(lactide-co-glycolic acid)-polyethylenimine (PLGA-PEI) (DPAP) delivery system, via a layer-by-layer synthesis method, using a water-in-oil-in-water double emulsion approach. The binding characteristics, targeting capability, biophysical properties, encapsulation efficiency, and drug release profile of the DPAP system were investigated under varying conditions of ionic strength, polymer composition and molecular weight (MW), and degree of PEGylation of the synthetic core. Experimental results showed increased drug release rate with increasing buffer ionic strength. DPAP particulate system obtained the highest drug release of 50% at day 9 at 1 M NaCl ionic strength. DPAP formulation, using PLGA 65:35 and PEI MW of ∼800 Da, demonstrated an encapsulation efficiency of 78.93%, and a loading capacity of 0.1605 mg bovine serum albumin per mg PLGA. DPAP (PLGA 65:35, PEI MW∼25 kDa) formulation showed a high release rate with a biphasic release profile. Experimental data depicted a lower targeting power and reduced drug release rate for the PEGylated DPAP formulations. The outcomes from the present study lay the foundation to optimize the performance of DPAP system as an effective synthetic drug carrier for targeted delivery.

Large-volume methacrylate monolith for plasmid purification. Process engineering approach to synthesis and application.

The extent of exothermicity associated with the construction of large-volume methacrylate monolithic columns has somewhat obstructed the realisation of large-scale rapid biomolecule purification especially for plasmid-based products which have proven to herald future trends in biotechnology. A novel synthesis technique via a heat expulsion mechanism was employed to prepare a 40 mL methacrylate monolith with a homogeneous radial pore structure along its thickness. Radial temperature gradient was recorded to be only 1.8 degrees C. Maximum radial temperature recorded at the centre of the monolith was 62.3 degrees C, which was only 2.3 degrees C higher than the actual polymerisation temperature. Pore characterisation of the monolithic polymer showed unimodal pore size distributions at different radial positions with an identical modal pore size of 400 nm. Chromatographic characterisation of the polymer after functionalisation with amino groups displayed a persistent dynamic binding capacity of 15.5 mg of plasmid DNA/mL. The maximum pressure drop recorded was only 0.12 MPa at a flow rate of 10 mL/min. The polymer demonstrated rapid separation ability by fractionating Escherichia coli DH5alpha-pUC19 clarified lysate in only 3 min after loading. The plasmid sample collected after the fast purification process was tested to be a homogeneous supercoiled plasmid with DNA electrophoresis and restriction analysis.

Phenolic Pollutant Uptake Properties of Molecular Templated Polymers Containing ß-Cyclodextrin.

Templated (T) and nontemplated (NT) cross-linked materials containing ß-cyclodextrin (ß-CD) and epichlorohydrin (EPH) were prepared at variable ß-CD-EPH ratios (1:15, 1:20, and 1:25) in the presence and absence of a molecular template (toluene). The structural characterization of the materials was carried out using spectroscopy (Fourier transform infrared, solids 13C NMR, and scanning electron microscopy) and thermogravimetric analysis. The adsorption properties were studied with phenol-based adsorbates (2,4,6-trinitrophenol (TNP) and p-nitrophenol (PNP)) at equilibrium and dynamic conditions. The monolayer adsorption capacity ( Qm) varied for the T-polymer/TNP systems ( Qm = 0.10-0.95 mmol/g) and NT-polymer/TNP systems ( Qm = 0.23-0.83 mmol/g). The range of Qm values for the T-polymer/PNP systems (0.26-0.62 mmol/g) exceeded that of the NT-polymer/PNP systems (0.23-0.40 mmol/g). The kinetic uptake profiles for the polymers and phenolphthalein were reliably described by the pseudo-first-order model. The ß-CD inclusion site accessibility for the polymers varied from 15 to 20%, according to the level of cross-linking, where the accessibility of the T-polymers exceeded that of the NT-polymers. The structural characterization and phenol adsorption properties provide complementary support for the role of tunable polymer morphology in adsorption processes. The role of two-site binding was demonstrated for linear and globular polymer materials according to their unique adsorption properties with phenols of variable size and hydrophile-lipophile character.

Process evaluation and in vitro selectivity analysis of aptamer-drug polymeric formulation for targeted pharmaceutical delivery.

Targeted drug delivery is a promising strategy to promote effective delivery of conventional and emerging pharmaceuticals. The emergence of aptamers as superior targeting ligands to direct active drug molecules specifically to desired malignant cells has created new opportunities to enhance disease therapies. The application of biodegradable polymers as delivery carriers to develop aptamer-navigated drug delivery system is a promising approach to effectively deliver desired drug dosages to target cells. This study reports the development of a layer-by-layer aptamer-mediated drug delivery system (DPAP) via a w/o/w double emulsion technique homogenized by ultrasonication or magnetic stirring. Experimental results showed no significant differences in the biophysical characteristics of DPAP nanoparticles generated using the two homogenization techniques. The DPAP formulation demonstrated a strong targeting performance and selectivity towards its target receptor molecules in the presence of non-targets. The DPAP formulation demonstrated a controlled and sustained drug release profile under the conditions of pH 7 and temperature 37 °C. Also, the drug release rate of DPAP formulation was successfully accelerated under an endosomal acidic condition of ∼pH 5.5, indicating the potential to enhance drug delivery within the endosomal micro-environment. The findings from this work are useful to understanding polymer-aptamer-drug relationship and their impact on developing effective targeted delivery systems.

Aptamer-Mediated Polymeric Vehicles for Enhanced Cell-Targeted Drug Delivery.

BACKGROUND: The search for smart delivery systems for enhanced pre-clinical and clinical pharmaceutical delivery and cell targeting continues to be a major biomedical research endeavor owing to differences in the physicochemical characteristics and physiological effects of drug molecules, and this affects the delivery mechanisms to elicit maximum therapeutic effects. Targeted drug delivery is a smart evolution essential to address major challenges associated with conventional drug delivery systems. These challenges mostly result in poor pharmacokinetics due to the inability of the active pharmaceutical ingredients to specifically act on malignant cells thus, causing poor therapeutic index and toxicity to surrounding normal cells. Aptamers are oligonucleotides with engineered affinities to bind specifically to their cognate targets. Aptamers have gained significant interests as effective targeting elements for enhanced therapeutic delivery as they can be generated to specifically bind to wide range of targets including proteins, peptides, ions, cells and tissues. Notwithstanding, effective delivery of aptamers as therapeutic vehicles is challenged by cell membrane electrostatic repulsion, endonuclease degradation, low pH cleavage, and binding conformation stability. OBJECTIVE: The application of molecularly engineered biodegradable and biocompatible polymeric particles with tunable features such as surface area and chemistry, particulate size distribution and toxicity creates opportunities to develop smart aptamer-mediated delivery systems for controlled drug release. RESULTS: This article discusses opportunities for particulate aptamer-drug formulations to advance current drug delivery modalities by navigating active ingredients through cellular and biomolecular traffic to target sites for sustained and controlled release at effective therapeutic dosages while minimizing systemic cytotoxic effects. CONCLUSION: A proposal for a novel drug-polymer-aptamer-polymer (DPAP) design of aptamer-drug formulation with stage-wise delivery mechanism is presented to illustrate the potential efficacy of aptamer- polymer cargos for enhanced cell targeting and drug delivery.

Biophysical characterization of layer-by-layer synthesis of aptamer-drug microparticles for enhanced cell targeting.

Targeted delivery of drug molecules to specific cells in mammalian systems demonstrates a great potential to enhance the efficacy of current pharmaceutical therapies. Conventional strategies for pharmaceutical delivery are often associated with poor therapeutic indices and high systemic cytotoxicity, and this result in poor disease suppression, low surviving rates, and potential contraindication of drug formulation. The emergence of aptamers has elicited new research interests into enhanced targeted drug delivery due to their unique characteristics as targeting elements. Aptamers can be engineered to bind to their cognate cellular targets with high affinity and specificity, and this is important to navigate active drug molecules and deliver sufficient dosage to targeted malignant cells. However, the targeting performance of aptamers can be impacted by several factors including endonuclease-mediated degradation, rapid renal filtration, biochemical complexation, and cell membrane electrostatic repulsion. This has subsequently led to the development of smart aptamer-immobilized biopolymer systems as delivery vehicles for controlled and sustained drug release to specific cells at effective therapeutic dosage and minimal systemic cytotoxicity. This article reports the synthesis and in vitro characterization of a novel multi-layer co-polymeric targeted drug delivery system based on drug-loaded PLGA-Aptamer-PEI (DPAP) formulation with a stage-wise delivery mechanism. A thrombin-specific DNA aptamer was used to develop the DPAP system while Bovine Serum Albumin (BSA) was used as a biopharmaceutical drug in the synthesis process by ultrasonication. Biophysical characterization of the DPAP system showed a spherical shaped particulate formulation with a unimodal particle size distribution of average size ∼0.685 µm and a zeta potential of +0.82 mV. The DPAP formulation showed a high encapsulation efficiency of 89.4 ± 3.6%, a loading capacity of 17.89 ± 0.72 mg BSA protein/100 mg PLGA polymeric particles, low cytotoxicity and a controlled drug release characteristics in 43 days. The results demonstrate a great promise in the development of DPAP formulation for enhanced in vivo cell targeting. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:249-261, 2018.

The suitability of DEAE-Cl active groups on customized poly(GMA-co-EDMA) continuous stationary phase for fast enzyme-free isolation of plasmid DNA.

The creation of a commercially viable and a large-scale purification process for plasmid DNA (pDNA) production requires a whole-systems continuous or semi-continuous purification strategy employing optimised stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian/bovine-derived enzymes and several built-in unit processes, thus affecting overall plasmid recovery, processing time and economics. Continuous stationary phases are known to offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance even at high flow rates. A monolithic stationary sorbent was synthesised via free radical liquid porogenic polymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with surface and pore characteristics tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with an amine active group for anion-exchange purification of pDNA from cleared lysate obtained from E. coli DH5alpha-pUC19 pellets in RNase/protease-free process. Characterization of the resin showed a unique porous material with 70% of the pores sizes above 300 nm. The final product isolated from anion-exchange purification in only 5 min was pure and homogenous supercoiled pDNA with no gDNA, RNA and protein contamination as confirmed with DNA electrophoresis, restriction analysis and SDS page. The resin showed a maximum binding capacity of 15.2 mg/mL and this capacity persisted after several applications of the resin. This technique is cGMP compatible and commercially viable for rapid isolation of pDNA.

Towards targeted cancer therapy: Aptamer or oncolytic virus?

Cancer is a leading cause of global mortality. Whilst anticancer awareness programs have increased significantly over the years, scientific research into the development of efficient and specific drugs to target cancerous cells for enhanced therapeutic effects has not received much clinical success. Chemotherapeutic agents are incapable of acting specifically on cancerous cells, thus causing low therapeutic effects accompanied by toxicity to surrounding normal tissues. The search for smart, highly specific and efficient cancer treatments and delivery systems continues to be a significant research endeavor. Targeted cancer therapy is an evolving treatment approach with great promise in enhancing the efficacy of cancer therapies via the delivery of therapeutic agents specifically to and into desired tumor cells using viral or non-viral targeting elements. Viral oncotherapy is an advanced cancer therapy based on the use of oncolytic viruses (OV) as elements to specifically target, replicate and kill malignant cancer cells selectively without affecting surrounding healthy cells. Aptamers, on the other hand, are non-viral targeting elements that are single-stranded nucleic acids with high specificity, selectivity and binding affinity towards their cognate targets. Aptamers have emerged as a new class of bioaffinity targeting elements can be generated and molecularly engineered to selectively bind to diverse targets including proteins, cells and tissues. This article discusses, comparatively, the potentials and impacts of both viral and aptamer-mediated targeted cancer therapies in advancing conventional drug delivery systems through enhanced target specificity, therapeutic payload, bioavailability of the therapeutic agents at the target sites whilst minimizing systemic cytotoxicity. This article emphasizes on effective site-directed targeting mechanisms and efficacy issues that impact on clinical applications.

Nano-formulations of drugs: Recent developments, impact and challenges.

Nano-formulations of medicinal drugs have attracted the interest of many researchers for drug delivery applications. These nano-formulations enhance the properties of conventional drugs and are specific to the targeted delivery site. Dendrimers, polymeric nanoparticles, liposomes, nano-emulsions and micelles are some of the nano-formulations that are gaining prominence in pharmaceutical industry for enhanced drug formulation. Wide varieties of synthesis methods are available for the preparation of nano-formulations to deliver drugs in biological system. The choice of synthesis methods depend on the size and shape of particulate formulation, biochemical properties of drug, and the targeted site. This article discusses recent developments in nano-formulation and the progressive impact on pharmaceutical research and industries. Additionally, process challenges relating to consistent generation of nano-formulations for drug delivery are discussed.

Development and characteristics of polymer monoliths for advanced LC bioscreening applications: A review.

Biomedical research advances over the past two decades in bioseparation science and engineering have led to the development of new adsorbent systems called monoliths, mostly as stationary supports for liquid chromatography (LC) applications. They are acknowledged to offer better mass transfer hydrodynamics than their particulate counterparts. Also, their architectural and morphological traits can be tailored in situ to meet the hydrodynamic size of molecules which include proteins, pDNA, cells and viral targets. This has enabled their development for a plethora of enhanced bioscreening applications including biosensing, biomolecular purification, concentration and separation, achieved through the introduction of specific functional moieties or ligands (such as triethylamine, N,N-dimethyl-N-dodecylamine, antibodies, enzymes and aptamers) into the molecular architecture of monoliths. Notwithstanding, the application of monoliths presents major material and bioprocess challenges. The relationship between in-process polymerisation characteristics and the physicochemical properties of monolith is critical to optimise chromatographic performance. There is also a need to develop theoretical models for non-invasive analyses and predictions. This review article therefore discusses in-process analytical conditions, functionalisation chemistries and ligands relevant to establish the characteristics of monoliths in order to facilitate a wide range of enhanced bioscreening applications. It gives emphasis to the development of functional polymethacrylate monoliths for microfluidic and preparative scale bio-applications.

NaCS-PDMDAAC immobilized cultivation of recombinant Dictyostelium discoideum for soluble human Fas ligand production.

Dictyostelium discoideum is a promising eukaryotic host for the expression of heterologous proteins requiring post-translational modifications. However, the dilute nature of D. discoideum cell culture limits applications for high value proteins production. D. discoideum cells, entrapped in sodium cellulose sulfate/poly-dimethyl-diallyl-ammonium chloride (NaCS-PDMDAAC) capsules were used for biosynthesis of the heterologous protein, soluble human Fas ligand (hFasL). Semi-continuous cultivations with capsules recycling were carried out in shake flasks. Also, a scaled-up cultivation of immobilized D. discoideum for hFasL production in a customized vitreous airlift bioreactor was conducted. The results show that NaCS-PDMDAAC capsules have desirable biophysical properties including biocompatibility with the D. discoideum cells and good mechanical stability throughout the duration of cultivation. A maximum cell density of 2.02 × 10(7) cells mL(-1) (equivalent to a maximum cell density of 2.22 × 10(8) cells mL(-1) in capsules) and a hFasL concentration of 130.40 µg L(-1) (equivalent to a hFasL concentration of 1434.40 µg L(-1) in capsules) were obtained in shake flask cultivation with capsules recycling. Also, a maximum cell density of 1.72 × 10(7) cells mL(-1) (equivalent to a maximum cell density of 1.89 × 10(8) cells mL(-1) in capsules) and a hFasL concentration of 106.10 µg L(-1) (equivalent to a hFasL concentration of 1167.10 µg L(-1) in capsules) were obtained after ∼170 h cultivation in the airlift bioreactor (with a working volume of 200 mL in a 315 mL bioreactor). As the article presents a premier work in the application of NaCS-PDMDAAC immobilized D. discoideum cells for the production of hFasL, more work is required to further optimize the system to generate higher cell densities and hFasL titers for large-scale applications.

Parametric Investigation of Batch Adsorption of Proteins onto Polymeric Particles.

BACKGROUND: Effective bimolecular adsorption of proteins onto solid matrices is characterized by in-depth understanding of the biophysical features essential to optimize the adsorption performance. RESULTS: The adsorption of bovine serum albumin (BSA) onto anion-exchange Q-sepharose solid particulate support was investigated in batch adsorption experiments. Adsorption kinetics and isotherms were developed as a function of key industrially relevant parameters such as polymer loading, stirring speed, buffer pH, protein concentration and the state of protein dispersion (solid/aqueous) in order to optimize binding performance and adsorption capacity. Experimental results showed that the first order rate constant is higher at higher stirring speed, higher polymer loading, and under alkaline conditions, with a corresponding increase in equilibrium adsorption capacity. Increasing the stirring speed and using aqueous dispersion protein system increased the adsorption rate, but the maximum protein adsorption was unaffected. Regardless of the stirring speed, the adsorption capacity of the polymer was 2.8 mg/ml. However, doubling the polymer loading increased the adsorption capacity to 9.4 mg/ml. CONCLUSIONS: The result demonstrates that there exists a minimum amount of polymer loading required to achieve maximum protein adsorption capacity under specific process conditions.

Molecular delivery of plasmids for genetic vaccination.

Plasmid vaccination is a smart gene delivery application mostly achieved through the utilisation of viral or copolymeric systems as surrogated carriers in micro or nano formulations. A common polymeric protocol for plasmid vaccine formulation, which as somewhat been successful, is via the complexation of the DNA molecules with a cationic polymer, and encapsulating in a vehicular carrier polymer. Even though plasmid vaccination research has not witnessed the much anticipated success, due a number of cellular and physicochemical reasons, application of copolymeric carriers with tight functionalities is a promising strategy to optimally deliver the DNA molecules; in view of the available chemistries and physical properties that could be tuned to enable enhanced targeted delivery, uptake and specific transfection. This also enables the targeting of specific epitopes and antigen presenting cells for the treatment of many pathogenic infections and cancer. This paper provides a brief critical review of the current state of plasmid vaccines formulation and molecular delivery with analysis of performance data obtained from clinical trials.

Extravasation of polymeric nanomedicines across tumor vasculature.

Tumor microvasculature is fraught with numerous physiological barriers which hinder the efficacy of anticancer agents. These barriers include chaotic blood supply, poor tumor vasculature permeability, limited transport across the interstitium due to high interstitial pressure and absence of lymphatic network. Abnormal microvasculature also leads to hypoxia and acidosis which limits effectiveness of chemotherapy. These barriers restrict drug or drug carrier extravasation which hampers tumor regression. Targeting key features of the tumor microenvironment such as tumor microvessels, interstitial hypertension and tumor pH is a promising approach to improving the efficacy of anticancer drugs. This review highlights the current knowledge on the distinct tumor microenvironment generated barriers which limit extravasation of drugs and focuses on modalities for overcoming these barriers using multi-functional polymeric carriers. Special attention is given to utilizing polymeric nanomedicines to facilitate extravasation of anticancer drugs for future cancer therapy.

Process optimisation for anion exchange monolithic chromatography of 4.2kbp plasmid vaccine (pcDNA3F).

Anion exchange monolithic chromatography is increasingly becoming a prominent tool for plasmid DNA purification but no generic protocol is available to purify all types of plasmid DNA. In this work, we established a simple framework and used it to specifically purify a plasmid DNA model from a clarified alkaline-lysed plasmid-containing cell lysate. The framework involved optimising ligand functionalisation temperature (30-80°C), mobile phase flow rate (0.1-1.8mL/min), monolith pore size (done by changing the porogen content in the polymerisation reaction by 50-80%), buffer pH (6-10), ionic strength of binding buffer (0.3-0.7M) and buffer gradient elution slope (1-10% buffer B/min). We concluded that preferential pcDNA3F adsorption and optimum resolution could be achieved within the tested conditions by loading the clarified cell lysate into 400nm pore size of monolith in 0.7M NaCl (pH 6) of binding buffer followed by increasing the NaCl concentration to 1.0M at 3%B/min.

Synthesis of biodegradable polymer-mesoporous silica composite microspheres for DNA prime-protein boost vaccination.

DNA vaccines or proteins are capable of inducing specific immunity; however, the translation to the clinic has generally been problematic, primarily due to the reduced magnitude of immune response and poor pharmacokinetics. Herein we demonstrate a composite microsphere formulation, composed of mesoporous silica spheres (MPS) and poly(D,L-lactide-co-glycolide) (PLGA), enables the controlled delivery of a prime-boost vaccine via the encapsulation of plasmid DNA (pDNA) and protein in different compartments. Method with modified dual-concentric-feeding needles attached to a 40 kHz ultrasonic atomizer was studied. These needles focus the flow of two different solutions, which passed through the ultrasonic atomizer. The process synthesis parameters, which are important to the scale-up of composite microspheres, were also studied. These parameters include polymer concentration, feed flowrate, and volumetric ratio of polymer and pDNA-PEI/MPS-BSA. This fabrication technique produced composite microspheres with mean D[4,3] ranging from 6 to 34 microm, depending upon the microsphere preparation. The resultant physical morphology of composite microspheres was largely influenced by the volumetric ratio of pDNA-PEI/MPS-BSA to polymer, and this was due to the precipitation of MPS at the surface of the microspheres. The encapsulation efficiencies were predominantly in the range of 93-98% for pDNA and 46-68% for MPS. In the in vitro studies, the pDNA and protein showed different release kinetics in a 40 day time frame. The dual-concentric-feeding in ultrasonic atomization was shown to have excellent reproducibility. It was concluded that this fabrication technique is an effective method to prepare formulations containing a heterologous prime-boost vaccine in a single delivery system.

Microparticle-mediated gene delivery for the enhanced expression of a 19-kDa fragment of merozoite surface protein 1 of Plasmodium falciparum.

The 19 kDa carboxyl-terminal fragment of merozoite surface protein 1 (MSP1(19)) is a major component of the invasion-inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles of malaria DNA vaccines encoding MSP1(19) is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate PLGA microparticles at a flow rate of 18 mL h(-1). High levels of gene expression and moderate cytotoxicity in COS-7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z-average hydrodynamic diameters of 1.50-2.10 microm and zeta potentials of 17.8-23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that PLGA-mediated microparticles can be employed as potential gene delivery systems to antigen-presenting cells in the prevention of malaria.