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

Inhalable spray-dried chondroitin sulphate microparticles: effect of different solvents on particle properties and drug activity

Spray-drying stands as one of the most used techniques to produce inhalable microparticles, but several parameters from both the process and the used materials affect the properties of the resulting microparticles. In this work, we describe the production of drug-loaded chondroitin sulphate microparticles by spray-drying, testing the effect of using different solvents during the process. Full characterisation of the polymer and of the aerodynamic properties of the obtained microparticles are provided envisaging an application in inhalable tuberculosis therapy. The spray-dried microparticles successfully associated two first-line antitubercular drugs (isoniazid and rifabutin) with satisfactory production yield (up to 85%) and drug association efficiency (60%-95%). Ethanol and HCl were tested as co-solvents to aid the solubilisation of rifabutin and microparticles produced with the former generally revealed the best features, presenting a better ability to sustainably release rifabutin. Moreover, these presented aerodynamic properties compatible with deep lung deposition, with an aerodynamic diameter around 4 μm and fine particle fraction of approximately 44%. Finally, it was further demonstrated that the antitubercular activity of the drugs remained unchanged after encapsulation independently of the used solvent.UID/Multi/04326/2019; SFRH/BD/52426/2013; ED481B 2018/071info:eu-repo/semantics/publishedVersio

Probing the luminal microenvironment of reconstituted epithelial microtissues.

Polymeric microparticles can serve as carriers or sensors to instruct or characterize tissue biology. However, incorporating microparticles into tissues for in vitro assays remains a challenge. We exploit three-dimensional cell-patterning technologies and directed epithelial self-organization to deliver microparticles to the lumen of reconstituted human intestinal microtissues. We also develop a novel pH-sensitive microsensor that can measure the luminal pH of reconstituted epithelial microtissues. These studies offer a novel approach for investigating luminal microenvironments and drug-delivery across epithelial barriers

Inhalable fucoidan microparticles combining two antitubercular drugs with potential application in pulmonary tuberculosis therapy

The pulmonary delivery of antitubercular drugs is a promising approach to treat lung tuberculosis. This strategy not only allows targeting the infected organ instantly, it can also reduce the systemic adverse effects of the antibiotics. In light of that, this work aimed at producing fucoidan-based inhalable microparticles that are able to associate a combination of two first-line antitubercular drugs in a single formulation. Fucoidan is a polysaccharide composed of chemical units that have been reported to be specifically recognised by alveolar macrophages (the hosts of Mycobacterium). Inhalable fucoidan microparticles were successfully produced, effectively associating isoniazid (97%) and rifabutin (95%) simultaneously. Furthermore, the produced microparticles presented adequate aerodynamic properties for pulmonary delivery with potential to reach the respiratory zone, with a mass median aerodynamic diameter (MMAD) between 3.6-3.9 mu m. The formulation evidenced no cytotoxic effects on lung epithelial cells (A549), although mild toxicity was observed on macrophage-differentiated THP-1 cells at the highest tested concentration (1 mg/mL). Fucoidan microparticles also exhibited a propensity to be captured by macrophages in a dose-dependent manner, as well as an ability to activate the target cells. Furthermore, drug-loaded microparticles effectively inhibited mycobacterial growth in vitro. Thus, the produced fucoidan microparticles are considered to hold potential as pulmonary delivery systems for the treatment of tuberculosis.Portuguese Foundation for Science and Technology [PTDC/DTP-FTO/0094/2012, UID/Multi/04326/2013, UID/BIM/04773/2013]; CAPES-Brazil [BEX 1168/13-4

Encapsulation of citrus by-product extracts by spray-drying and freeze-drying using combinations of maltodextrin with soybean protein and ι-carrageenan

The effect of different combinations of maltodextrin (MD) coating agents (MD, MD + soybean protein, and MD + ι-carrageenan) on the encapsulation of lemon by-product aqueous extracts using freeze-drying and spray-drying were investigated. The total phenolic content (TPC), total flavonoid content (TFC), and ferric ion reducing antioxidant power (FRAP) of the microparticles were evaluated. Freeze-drying with the mixture of MD + soybean protein resulted in the highest retention of TPC, TFC, and FRAP (1.66 ± 0.02 mg GAE/g d.b., 0.43 ± 0.02 mg CE/g d.b., and 3.70 ± 0.05 mM TE/g, respectively). Freeze-drying resulted in microparticles with lower moisture content (MC) and water activity (aw) than those produced by spray-drying. Specifically, the MC and aw of the microparticles produced by freeze-drying ranged from 1.15 to 2.15% and 0.13 to 0.14, respectively, while the MC and aw of the microparticles produced by spray-drying ranged from 6.06% to 6.60% and 0.33 to 0.40, respectively. Scanning electron microscopy revealed that spray-drying resulted in the formation of spherical particles of different sizes regardless of the type of coating agent. Although freeze-drying resulted in microparticles with amorphous glassy shapes, the mixture of MD + soybean protein resulted in the formation of spherical porous particles. X-ray diffraction revealed a low degree of crystallinity for the samples produced by both techniques.</p

A new way of valorizing biomaterials: the use of sunflower protein for 1 a-tocopherol microencapsulation

Biopolymer based microparticles were efficiently prepared from sunflower protein (SP) wall material and a-tocopherol (T) active core using a spray-drying technique. Protein enzymatic hydrolysis and/or N-acylation were carried out to make some structural modifications to the vegetable protein. Native and hydrolyzed SP were characterized by Asymmetrical Flow Field-Flow Fractionation (AsFlFFF). Results of AsFlFFF confirmed that size of proteinic macromolecules was influenced by degree of hydrolysis. The effect of protein modifications and the influence of wall/core ratio on both emulsions and microparticle properties were evaluated. Concerning emulsion properties, enzymatic hydrolysis involved a decrease in viscosity, whereas acylation did not significantly affect emulsion droplet size and viscosity. Microparticles obtained with hydrolyzed SP wall material showed lower retention efficiency (RE) than native SP microparticles (62-80% and 93% respectively). Conversely, acylation of both hydrolyzed SP and native SP allowed a higher RE to be reached (up to 100%). Increasing T concentration increased emulsion viscosity, emulsion droplet size, microparticle size, and enhanced RE. These results demonstrated the feasibility of high loaded (up to 79.2% T) microparticles

Tenocytic induction of stem cells from bone marrow on polymeric microparticles for a new concept of tendon regenerative prosthesis

A new concept of a regenerative and resorbable prosthesis for tendon and ligament has been developed. The prosthesis consists of a poly-lactide acid (PLA) braid, microparticles in its interior serving as cell carriers, and a surface non-adherent coating. The aim of this study is to select the most suitable support, microparticles of poly-L-lactide (PLLA) or chitosan (CHT), for carrying the cells inside the hollow PLA braid. Microparticles of these polymers were manufactured and blended with microparticles of hyaluronic acid (HA). All of them were physically and biologically characterized. Cell viability, morphology and proliferation of human mesenchymal stem cells (hMSCs) on the different supports were evaluated and compared, revealing that PLLA microparticles were the most appropriate to be used as injectable cell-carrier. Finally, hMSCs differentiation into tenocytes was carried out on PLLA microparticles using bone morphogenetic protein-12 (BMP-12) and a mixture of transforming growth factor-β1 (TGF-β1) and insulin-like growth factor1 (IGF-1). Cell morphology was analyzed by electronic and confocal microscopy and cell differentiation was evaluated immunocytochemically for the presence of type I collagen and tenomodulin. Besides, the tenomodulin and decorin gene expression were measured by real-time quantitative polymerase chain reaction (RT-qPCR). Our results showed that the medium supplemented with BMP-12 promoted higher expression of tenomodulin and decorin, both of them differentiation markers of tenocytes. This approach might be relevant to future tissue engineering applications in reconstruction of tendon and ligament defects. Authors acknowledge support of the Spanish Instituto de Salud Carlos III through CIBERbbn and the Spanish Network on Cell Therapy (Red TerCel) initiatives.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

PLA Microparticles for Pulmonary Delivery of AntiTB drugs: Biodistribution study

A dry powder inhalable (DPI) microparticles comprising anti-tuberculosis drugs incorporated in biodegradable polymers was developed for the treatment of pulmonary tuberculosis (P. Muttil _et al_. 2007). Poly L-lactic acid (PLA) microparticles incorporating a high payload of rifabutin and isoniazid were fabricated by spray drying (Buchi 190). Microparticles were composed of PLA and the drugs (rifabutin and isoniazid) at a 2:1:1 weight ratio. Microparticles of desired high encapsulation efficiency and sustained release characteristics were produced having a diameter range of 2-10 &#xb5;m (Malvern Mastersizer 2000). Differential scanning calorimetry (DSC) was carried out to study drug polymer interaction. The time course of tissue biodistribution following a single inhalation dose of microparticles was evaluated. &#xd;&#xa;Thirty-two BALB/c mice were divided into groups of four and administered the DPI using an in-house (nose only) apparatus (Kaur _et al_. 2008; Verma _et al_. 2008). A validated HPLC method was used for determination of rifabutin and isoniazid in the lungs (target organ), liver and kidneys (major sites of toxicity) at different time-points after inhalation. A comparison was made with mice receiving free drugs (intravenous) at equivalent doses. Deposition of microparticles in lungs of mice following aerosolization was also evaluated. Pharmacokinetic parameters in different organs were calculated using WinNonlin software version 5.2. Area under the concentration-time curve observed (AUC~obs~), C~max~, half-life (t~&#xbd;~) and clearance (CL) in lungs following inhalation /intravenous administration were:&#xd;&#xa;*Rifabutin*: AUC~obs~-96h= 1697.39 &#xb1;154.67 (187.63 &#xb1;23.93) &#xb5;g/ml^-1^hr^-1^; C~max~ = 33.42&#xb1;3.80 (4.17&#xb1;0.31) &#xb5;g.ml^-1^; t~&#xbd;~= 78.08&#xb1;9.42 (34.00 &#xb1;3.31) and Cl= 1.16&#xb1;.22 (0.68 &#xb1;0.45) ml.h^-1^.&#xd;&#xa;*Isoniazid*: AUC~obs~-24h= 566.31&#xb1;123.96 (99.85 &#xb1;14.24) &#xb5;g/ml^-1^hr^-1^; Cmax= 24.02&#xb1;1.71 (8.16&#xb1;0.93) &#xb5;g.ml^-1^; t~&#xbd;~= 25.88&#xb1;12.16 (6.45&#xb1;3.24) h; and Cl= 5.47&#xb1;1.30 (0.96&#xb1;0.14) ml.h^-1^.&#xd;&#xa;The relative bioavailability of both drugs incorporated in microparticles was significantly higher compared with free drugs. Peak levels of isoniazid and rifabutin in lungs (target organ) were much higher than those in the liver and kidney of mice in case of inhalation as compared to intravenous administration. Inhalation of microparticles resulted in targeting both drugs to the lungs, with the effect being more pronounced in the case of rifabutin than isoniazid. High and prolonged drug concentrations and increased AUC values (~9-fold and ~6 fold increase of rifabutin and isoniazid in case of lungs) with respect to free drugs were observed. Significant decrease in drug concentration was found in the liver and kidneys. Drug levels were maintained above the minimum inhibitory concentration (MIC) in organs through out the study after administration of encapsulated drugs. Based on favorable biodistribution kinetics, these microparticles hold great potential in reducing dosing frequency and toxicity of antituberculosis drugs.&#xd;&#xa