Pulsatile Protein Release from Monodisperse Liquid-Core Microcapsules of Controllable Shell Thickness

Purpose

Pulsatile delivery of proteins, in which release occurs over a short time after a period of little or no release, is desirable for many applications. This paper investigates the effect of biodegradable polymer shell thickness on pulsatile protein release from biodegradable polymer microcapsules.

Methods

Using precision particle fabrication (PPF) technology, monodisperse microcapsules were fabricated encapsulating bovine serum albumin (BSA) in a liquid core surrounded by a drug-free poly(lactide-co-glycolide) (PLG) shell of uniform, controlled thickness from 14 to 19 μm.

Results

When using high molecular weight PLG (Mw 88 kDa), microparticles exhibited the desired core-shell structure with high BSA loading and encapsulation efficiency (55–65%). These particles exhibited very slow release of BSA for several weeks followed by rapid release of 80–90% of the encapsulated BSA within 7 days. Importantly, with increasing shell thickness the starting time of the pulsatile release could be controlled from 25 to 35 days.

Conclusions

Biodegradable polymer microcapsules with precisely controlled shell thickness provide pulsatile release with enhanced control of release profiles.     

Characterization of Solid Maltose Microneedles and their Use for Transdermal Delivery

Purpose To characterize solid maltose microneedles and assess their ability to increase transdermal drug delivery. Materials and Methods Microneedles and microchannels were characterized using methylene blue staining and scanning electron microscopy. Diffusion pattern of calcein was observed using confocal scanning laser microscopy. Transepidermal water loss (TEWL) measurements were made to study the skin barrier recovery after treatment. Uniformity in calcein uptake by the pores was characterized and percutaneous penetration of nicardipine hydrochloride (NH) was studied in vitro and in vivo across hairless rat skin. Results Microneedles were measured to be 508.46 ± 9.32 μm long with a radius of curvature of 3 μm at the tip. They penetrated the skin while creating microchannels measuring about 55.42 ± 8.66 μm in diameter. Microchannels were visualized by methylene blue staining. Pretreatment with microneedles resulted in the migration of calcein into the microchannels. TEWL increased after pretreatment and uptake of calcein by the pores was uniform as measured by the pore permeability index values. NH in vitro transport across skin increased significantly after pretreatment (flux 7.05 μg/cm²/h) as compared to the untreated skin (flux 1.72 μg/cm²/h) and the enhanced delivery was also demonstrated in vivo in hairless rats. Conclusion Maltose microneedles were characterized and shown to create microchannels in the skin, which were also characterized and shown to improve the transdermal delivery of NH.     

Co-encapsulation of dexamethasone 21-acetate and SPIONs into biodegradable polymeric microparticles designed for intra-articular delivery

Objective: Intra-articular drug delivery systems still suffer from too short-lasting effects. Magnetic particles retained in the joint using an external magnetic field might prolong the local release of an anti-inflammatory drug. For the purpose, superparamagnetic iron oxide nanoparticles (SPIONs) and dexamethasone 21-acetate (DXM) were co-encapsulated into biodegradable microparticles.

Methods: Poly(D,L-lactide-co-glycolide) microparticles embedding both SPIONs and DXM were prepared by a double emulsion technique. The formulation was optimized in two steps, a screening design and a full factorial design, aiming at 10-μm particle diameter and high DXM encapsulation efficacy.

Results: The most significant parameters were the polymer concentration, the stirring speed during solvent extraction and the extractive volume. Increasing the polymer concentration from 200 to 300 mg ml^?1, both the microparticle mean diameter and the DXM encapsulation efficacy increased up to 12 μm and 90%, respectively. The microparticles could be retained with an external magnet of 0.8 T placed at 3 mm. Faster DXM release was obtained for smaller microparticles.

Conclusion: The experimental set-up offered the tools for tailoring a formulation with magnetic retention properties and DXM release patterns corresponding to the required specifications for intra-articular administration.     

Cetirizine dihydrochloride loaded microparticles design using ionotropic cross-linked chitosan nanoparticles by spray-drying method

To control the release rate and mask the bitter taste, cetirizine dihydrochloride (CedH) was entrapped within chitosan nanoparticles (CS-NPs) using an ionotropic gelation process, followed by microencapsulation to produce CS matrix microparticles using a spray-drying method. The aqueous colloidal CS-NPs dispersions with a drug encapsulation efficiency (EE) of <15%, were then spray dried to produce a powdered nanoparticles-in-microparticles system with an EE of >70%. The resultant spherical CS microparticles had a smooth surface, were free of organic solvent residue and showed a diameter range of 0.5∼5 μm. The in vitro drug release properties of CedH encapsulated microparticles showed an initial burst effect during the first 2 h. Drug release from the matrix CS microparticles could be retarded by the crosslinking agent pentasodium tripolyphosphate or the wall material. The technique of ‘ionotropic gelation’ combined with ‘spray-drying’ could be applicable for preparation of CS nanoparticlesin-microparticles drug delivery systems. CS-NPs based microparticles might provide a potential micro-carrier for oral administration of the freely water-soluble drug — CedH.     

Adsorption of a Novel Recombinant Glycoprotein from HIV (Env gp120dV2 SF162) to Anionic PLG Microparticles Retains the Structural Integrity of the Protein,Whereas Encapsulation in PLG Microparticles Does Not

No HeadingPurpose. To evaluate the delivery of a novel HIV-1 antigen (gp120dV2 SF162) by surface adsorption or encapsulation within polylactide-co-glycolide microparticles and to compare both the formulations for their ability to preserve functional activity as measured by binding to soluble CD4.Methods. Poly(lactide-co-glycolide) microparticles were synthesized by a water-in-oil-in-water (w/o/w) emulsification method in the presence of the anionic surfactant dioctylsulfosuccinate (DSS) or polyvinyl alcohol. The HIV envelope glyocoprotein was adsorbed and encapsulated in the PLG particles. Binding efficiency and burst release measured to determine adsorption characteristics. The ability to bind CD4 was assayed to measure the functional integrity of gp120dV2 following different formulation processes.Results. Protein (antigen) binding to PLG microparticles was influenced by both electrostatic interaction and other mechanisms such as hydrophobic attraction and structural accommodation of the polymer and biomolecule. The functional activity as measured by the ability of gp120dV2 to bind CD4 was maintained by adsorption onto anionic microparticles but drastically reduced by encapsulation.Conclusions. The antigen on the adsorbed PLG formulation maintained its binding ability to soluble CD4 in comparison to encapsulation, demonstrating the feasibility of using these novel anionic microparticles as a potential vaccine delivery system.     

Controlled Release from Coated Polymer Microparticles Embedded in Tissue-engineered Scaffolds

The controlled release of proteins in tissue-engineered implants is being examined with the potential application to improve vascularization and hasten tissue growth. Bovine serum albumin (BSA), was encapsulated within poly(D,L-lactic-co-glycolic acid) [PLGA] microparticles. The microparticles were coated with poly(vinyl alcohol) and incorporated into PLGA tissue-engineered scaffolds during fabrication. The release of BSA from PLGA microparticles, coated PLGA microparticles, and microparticles embedded in a porous PLGA scaffold was measured. We have developed a novel approach that will permit incorporation of coated polymeric microparticles during PLGA scaffold fabrication. Growth factors or drugs could be incorporated into the microparticles resulting in a long-term, controlled release.     

Collagen-coated polycaprolactone microparticles as a controlled drug delivery system

Objective: Polycaprolactone (PCL) microparticles coated with acetylated collagen have been assessed for use as a controlled drug delivery system.

Method: The surface morphology, drug encapsulation and release profile of PCL microparticles and collagen-coated PCL microparticles containing doxycycline hydrochloride (DH) have been investigated in order to develop a controlled release system which would in addition act as a scaffold for cell attachment. PCL microparticles were prepared by emulsion solvent evaporation technique and loaded with DH. Since the encapsulation was found to be low, PCL microparticles were coated with acetylated collagen containing DH, to increase the drug availability. Collagen was modified by acetylation to shift its isoelectric point and to have acetylated collagen solution at pH 7.0. The microparticles were characterized using a scanning electron microscope (SEM) and the in vitro drug release profile was determined using HPLC.

Results: Uniform sized (～1000 nm) PCL microparticles were prepared using 4% PVA in the external water phase. Acetylated collagen at pH 7.0 was coated onto the PCL microparticles. This resulted in microparticles of uniform size at neutral pH. PCL acts as a support for collagen which acts as a scaffold for cell attachment. In vitro drug release studies show that collagen-coated PCL microparticle is a promising candidate for controlled drug delivery system having release duration of over 10 days. In vitro fibroblast culture studies reveal that collagen is a good substrate for cell attachment and would provide a stable environment for cell proliferation and regeneration. Thus, this system would be ideal for a short-term drug delivery to create an aseptic environment where cells can adhere and proliferate to regenerate the site.     

Release optimization of epidermal growth factor from PLGA microparticles

Abstract

The objective of this study was to prepare poly lactic-co-glycolic acid (PLGA)-based microparticles as potential carriers for recombinant human epidermal growth factor (rhEGF). In order to optimize characteristic parameters of protein-loaded microspheres, bovine serum albumin (BSA) was selected as the model protein. To reduce burst release as a common problem of microspheres, a proper alteration in the particle composition was used, such as addition of poly vinyl alcohol and changes in initial drug loading. The effects of these parameters on particle size, encapsulation efficiency and in vitro release kinetics of BSA in PLGA microspheres were investigated using a Box–Behnken response surface methodology. The biological activity of the released rhEGF was assessed using human skin fibroblasts cell proliferation assay. The prepared rhEGF-loaded microspheres had an average size of 6.44?±?2.45?µm, encapsulation efficiency of 97.04?±?1.13%, burst release of 13.06?±?1.35% and cumulative release of 22.56?±?2.41%. The proliferation of human skin fibroblast cells cultivated with rhEGF releasate of microspheres was similar to that of pure rhEGF, indicating the biological activity of released protein confirming the stability of rhEGF during microsphere preparation. These results are in agreement with the purpose of our study to prepare rhEGF-entrapped PLGA microparticles with optimized characteristics.     

Pharmacodynamic Evaluation of Oral Estradiol Nanoparticles in Estrogen Deficient (Ovariectomized) High-Fat Diet Induced Hyperlipidemic Rat Model

Purpose  It is believed that estrogen deficiency contributes importantly to the pathogenesis of menopausal metabolic syndrome and symptoms can be ameliorated with estradiol therapy. The present study reports efficacy of 17-β estradiol encapsulated nanoparticles in treating the postmenopausal dyslipidemic condition. Materials and Methods  Estradiol encapsulated poly(lactide-co-glycolide) (PLGA) nanoparticles were prepared by emulsion–diffusion–evaporation method and evaluated in estrogen deficient (ovariectomized) high fat diet induced hyperlipidemic rat model. Results  The results obtained showed that estradiol nanoparticles were equally/more effective in treatment of estrogen deficient hyperlipidemic conditions at three times reduced dose and frequency in comparison to that of drug suspension administered orally. Conclusion  Together, these results demonstrate the ability of nanoparticles in improving oral bioavailability/efficacy of estradiol.     

日夜两用生长激素微针贴片的制备及体外评价

目的 制备日夜两用生长激素微针贴片，模拟人体生理状态下内源性生长激素分泌的昼夜差异，实现生长激素给药时间和用量的优化，同时有效减轻皮下注射给药疼痛感，提高患者使用依从性。方法 采用铸模法制备微针贴片，通过光学显微镜和扫描电子显微镜观察微针表面形貌。经体外释放试验确定含生长激素微针制备的最佳工艺条件，包括优化紫外交联时间和交联剂含量。通过微针力学强度测试和体外透皮试验验证微针贴片有效穿透皮肤的可行性，通过圆二色光谱测定药物的稳定性。通过调试负载不同剂量生长激素制备日用和夜用微针贴片。结果 在显微镜下观察到微针排列整齐，针体完整、尖锐，微针在药物释放前后形貌无明显差异。工艺优化结果表明，当紫外交联时间为7 min，交联剂用量为1.5%时，微针贴片可以有效穿透离体大鼠皮肤，同时实现了生长激素在12 h内稳定释放，且微针释放出的蛋白药物构象无明显变化。通过在针体中负载不同剂量生长激素，制备了日用和夜用生长激素微针贴片。结论 本研究制备的微针能够顺应在生理状态下生长激素分泌的日夜差异，实现了适宜时间释放适量生长激素的目标，未来可进一步优化微针药物负载量以满足不同患者的实际使用需求，以期实现个体化治疗。     

Fabrication and characterisation of self-applicating heparin sodium microneedle patches

Abstract

The aim of this study was to develop heparin sodium loaded microneedle patches using different compositions of polyvinyl alcohol polymer and sorbitol. A vacuum micromolding technique was used to fabricate microneedle patches while heparin sodium was loaded into needle tips. Physical features of patches were evaluated by measuring thickness, width, folding endurance and swelling percentage. Patches were also characterised by optical microscopy and scanning electron microscopy to determine the microneedle length and surface morphologies. A preliminary assessment of the microneedle performance was studied by examining the in-vitro insertion to the parafilm and recording the in-vitro drug release profile. In-vivo activity of patches was confirmed by measuring activated partial thromboplastin time and histological examination of the micropierced skin tissues. Prepared patches were clear, smooth; uniform in appearance; with sharp pointed microprojections and remained intact after 1000 folding. The microneedles were stiffer in nature, as they reproduce microcavities in the parafilm membrane following hand pushing without any structural loss. Insertion study results showed successful insertion of microneedles into the parafilm. Disrupted stratum corneum evident from histological examination confirmed successful insertion of the microneedle without affecting the vasculature. In-vitro release study confirmed ～92% release of the loaded drug within 120?min. A significant prolongation of activated partial thromboplastin time (4 folds as compared to negative control) was recorded following the application of heparin sodium loaded microneedle patch onto rabbit skin. In conclusion microneedles are a valuable drug delivery system, benefiting the patients with minimal skin invasion and also allowing self-administration of heparin sodium in a sustained release manner for the management of chronic ailments.     

Fabrication of Dissolving Polymer Microneedles for Controlled Drug Encapsulation and Delivery: Bubble and Pedestal Microneedle Designs

Dissolving microneedle patches offer promise as a simple, minimally invasive method of drug and vaccine delivery to the skin that avoids the need for hypodermic needles. However, it can be difficult to control the amount and localization of drug within microneedles. In this study, we developed novel microneedle designs to improve control of drug encapsulation and delivery using dissolving microneedles by (i) localizing drug in the microneedle tip, (ii) increasing the amount of drug loaded in microneedles while minimizing wastage, and (iii) inserting microneedles more fully into the skin. Localization of our model drug, sulforhodamine B in the microneedle tip by either casting a highly concentrated polymer solution as the needle matrix or incorporating an air bubble at the base of the microneedle achieved approximately 80% delivery within 10 min compared to 20% delivery achieved by the microneedles encapsulating nonlocalized drug. As another approach, a pedestal was introduced to elevate each microneedle for more complete insertion into the skin and to increase its drug loading capacity by threefold from 0.018 to 0.053 μL per needle. Altogether, these novel microneedle designs provide a new set of tools to fabricate dissolving polymer microneedles with improved control over drug encapsulation, loading, and delivery.     

Preparation and evaluation of polymer based microcarriers for all-trans-retinoic acid

Polymeric biomaterials are being investigated for the last several years because of their controllable properties. In this study, it was aimed to prepare and evaluate the atRA carrying system using Poly(lactide-co-glycolide) (PLGA). Microparticles were characterized in terms of morphology and encapsulation efficiency. Release studies were performed for the evaluation of drug release rates. Cytotoxicity tests were implemented on MCF-7 Human breast cancer cell line for the investigation of drug and polymer toxicity. The microparticles were found smooth and spherical in shape. However, as the loaded drug amount increased, the sizes of microparticles also increased and the size distribution became less uniform. The sizes of atRA-loaded microparticles ranged between 1–10 µm. The encapsulation efficiency of atRetinoic acid (all-trans-Retinoic acid) was achieved approximately %90. Approximately, 45% of atRA was released from atRA-loaded microparticles by the end of 4–5 days. Cell growth inhibition was observed after 4 days of incubation of cells with PLGA microparticles.     

Synchrotron radiation-based Fourier-transform infrared spectromicroscopy for characterization of the protein/peptide distribution in single microspheres

The present study establishes a visualization method for the measurement of the distribution and localization of protein/peptide constituents within a single poly-lactide-co-glycolide (PLGA) microsphere using synchrotron radiation–based Fourier-transform infrared spectromicroscopy (SR-FTIR). The representative infrared wavenumbers specific for protein/peptide (Exenatide) and excipient (PLGA) were identified and chemical maps at the single microsphere level were generated by measuring and plotting the intensity of these specific bands. For quantitative analysis of the distribution within microspheres, Matlab software was used to transform the map file into a 3D matrix and the matrix values specific for the drug and excipient were extracted. Comparison of the normalized SR-FTIR maps of PLGA and Exenatide indicated that PLGA was uniformly distributed, while Exenatide was relatively non-uniformly distributed in the microspheres. In conclusion, SR-FTIR is a rapid, nondestructive and sensitive detection technology to provide the distribution of chemical constituents and functional groups in microparticles and microspheres.KEY WORDS: Fourier-transform infrared spectromicroscopy, Protein distribution, Microsphere, Exenatide, PLGA     

Visual Evidence of Acidic Environment Within Degrading Poly(lactic-co-glycolic acid) (PLGA) Microspheres

Purpose. In the past decade, biodegradable polymers have becomethe materials of choice for a variety of biomaterials applications. Inparticular, poly(lactic-co-glycolic acid) (PLGA) microspheres havebeen extensively studied for controlled-release drug delivery. However,degradation of the polymer generates acidic monomers, andacidification of the inner polymer environment is a central issue in thedevelopment of these devices for drug delivery. Methods. To quantitatively determine the intrapolymer acidity, weentrapped pH-sensitive fluorescent dyes (conjugated to 10,000 Dadextrans) within the microspheres and imaged them with confocalfluorescence microscopy. The technique allows visualization of thespatial and temporal distribution of pH within the degradingmicrospheres (1). Results. Our experiments show the formation of a very acidicenvironment within the particles with the minimum pH as low as 1.5. Conclusions. The images show a pH gradient, with the most acidicenvironment at the center of the spheres and higher pH near the edges,which is characteristic of diffusion-controlled release of the acidicdegradation products.     

Preliminary Assessment of the Immune Response to Olea europaea Pollen Extracts Encapsulated into PLGA Microspheres

In this study, poly (d,l-lactide-co-glycolide) (PLGA) microspheres encapsulating Olea europaea pollen extracts were prepared by using the double emulsion (w/o/w) based on a solvent evaporation/extraction method. The resulting microspheres were 1.93 μm in size. The total allergen loading and surface-associated allergen were 8 and 0.64%, respectively. The release of the allergen from the microspheres showed a biphasic profile with an initial burst release followed by a sustained release phase. Finally, the polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the encapsulation process does not affect the stability of the protein. We describe here some preliminary observations concerning the use of these microspheres as parenteral antigen delivery systems for immunization with O. europaea pollen extracts, in a small animal model, the mouse.     

Preparation of aminoglycoside-loaded chitosan nanoparticles using dextran sulphate as a counterion

The objective of this study was to apply the specific acid-catalysed hydrolysis of ethyl acetate to completing solvent extraction during an emulsion-based microencapsulation process. The dispersed phase consisting of poly-D,L-lactide-co-glycolide and ethyl acetate was emulsified in an acid catalyst containing aqueous phase. Catalytic hydrolysis of ethyl acetate led to its continual leaching from the dispersed phase of the emulsion, thereby triggering microsphere hardening with high efficiency. Ketoprofen was successfully encapsulated into microspheres via this technique, and liquid chromatography–mass spectrometry showed that its structural integrity was preserved during microencapsulation. Compared to typical solvent extraction approaches, the acid-catalysis technique helped minimize the consumption of a quench liquid. Also, the resultant microspheres displayed excellent dispersibility and decreased propensity for aggregation. Furthermore, the new method provided better drug encapsulation efficiency and lower levels of residual ethyl acetate in microspheres. In conclusion, the acid-catalysis approach had great potential for the preparation of versatile microspheres and nanoparticles.     

Assessment of formulated amodiaquine microparticles in Leishmania donovani infected rats

The aim of this study was to formulate, characterise and evaluate the activity of amodiaquine microparticles against Leishmania donovani. Microparticles were formulated by encapsulating the drug in bovine serum albumin using the spray-dryer method. The microparticles were evaluated for size, zeta potential, drug content, encapsulation efficiency and in vitro release profile. The size range of the microparticles formulated was between 1.9 and 10?μm with an average zeta potential of ?25.5?mV. Of the expected 20% drug loading, an average of 18.27% was obtained giving an encapsulation efficiency of 91.35%. Pharmacokinetic profile of amodiaquine improved with microencapsulation of the drug with C_max, AUC_0→48 and t_1//2 all significantly higher than amodiaquine solution. Amodiaquine microparticles showed an overall higher bioavailability and hence were more effective in eliminating intra-tissue parasites than the drug solution. It would therefore be expected that the formulated microparticles will be more effective in treating visceral leishmaniasis.     

Paper 2. Epigallocatechin Gallate and Tannic Acid Based Formulations of Finasteride for Dermal Administration and Chemoembolization

Finasteride is used to treat benign prostatic hyperplasia (BPH) and pattern hair loss (androgenetic alopecia or APA). The local administration of formulations with increased solubility and controlled release of finasteride are proposed using gallate-containing compositions within embolic microparticles or paste. Finasteride solubility in either epigallocatechin gallate (EGCG) or tannic acid (TA) solutions was assessed using HPLC. Poly(dl-lactide-co-glycolide) (PLGA) or poly(methylmethacrylate) (PMMA) microspheres (100–400 μm) containing finasteride and EGCG or TA were effectively manufactured. Embolic particles were loaded with finasteride/EGCG/TA. Dermal uptake of TA/EGCG/finasteride topical compositions was measured in pig skin. The solubility of finasteride was dramatically increased using EGCG- or TA-based compositions. Finasteride loaded microspheres released over two months which was increased by EGCG or TA inclusion. Embolic particles soaked up finasteride and EGCG or TA and released the encapsulated drug over two weeks. Dermal uptake of finasteride from EGCG- or TA-based formulations was enhanced between 10 and 50 fold in layers as deep as 500 μm when compared to a generic control formulation. Gallate-based formulations of finasteride increase drug solubility and allow for effective release of the drug from embolic formulations. Paste or powder EGCG- or TA-based formulations of finasteride greatly increase dermal penetration of the drug.     

Injectable hybrid delivery system composed of gellan gum,nanoparticles and gentamicin for the localized treatment of bone infections

ABSTRACT

Objectives: Bone infections are treated with antibiotics administered intravenously, antibiotic-releasing bone cements or collagen sponges placed directly in the infected area. These approaches render limited effectiveness due to the lack of site specificity and invasiveness of implanting cements and sponges. To address these limitations, we developed a novel polysaccharide hydrogel-based injectable system that enables controlled delivery of gentamicin (GENT). Its advantages are minimal invasiveness, and localized and finely regulated release of the drug.

Methods: GENT was incorporated both directly within the gellan gum hydrogel and into poly(L-lactide-co-glycolide) nanoparticles embedded into the hydrogel.

Results: We confirmed the injectability of the system and measured extrusion force was 15.6 ± 1.0 N, which is suitable for injections. The system set properly after the injection as shown by rheological measurements. Desired burst release of the drug was observed within the first 12 h and the dose reached ~27% of total GENT. Subsequently, GENT was released gradually and sustainably: ~60% of initial dose within 90 days. In vitro studies confirmed antimicrobial activity of the system against Staphylococcus spp. and cytocompatibility with osteoblast-like cells.

Conclusions: Developed injectable system enables minimally invasive, local and sustained delivery of the pharmaceutically relevant doses of GENT to combat bone infections.