Polyvinylpyrrolidone microneedles enable delivery of intact proteins for diagnostic and therapeutic applications

We present a method of fabricating microneedles from polyvinylpyrrolidone (PVP) that enables delivery of intact proteins (or peptides) to the dermal layers of the skin. PVP is known to self-assemble into branched hollow fibers in aqueous and alcoholic solutions; we utilized this property to develop dissolvable patches of microneedles. Proteins were dissolved in concentrated PVP solution in both alcohol and water, poured into polydimethylsiloxane templates shaped as microneedles and, upon evaporation of solvent, formed into concentric, fibrous, layered structures. This approach of making PVP microneedles overcomes problems in dosage, uniform delivery and stability of protein formulation as compared to protein-coated metallic microneedles or photopolymerized PVP microneedles. Here we characterize the PVP microneedles and measure the delivery of proteins into skin. We show that our method of fabrication preserves the protein conformation. These microneedles can serve as a broadly useful platform for delivering protein antigens and therapeutic proteins to the skin, for example for allergen skin testing or immunotherapy.     

Impact of physical properties of formulations on bioavailability of active substance: current and novel drugs with cyclosporine

During the long period of cyclosporine-containing dosage forms development a lot of significant findings have been done especially in the field of drug delivery systems. Currently available drugs are based, from technological point of view, on self-dispersible drug delivery systems, which contain cyclosporine solved in pharmaceutically acceptable vehicle. One can find difference among particular systems: (a) at particle size distribution after dispergation; (b) at composition; and (c) at bioavailability of cyclosporine. As far as improvement of bioavailability between original brand leader formulation Sandimmune and recent brand leader formulation Neoral was followed by significant improvement in particle size distribution, it was generally assumed that the reason for this improvement have been found. Several other formulations, e.g. Consupren or SangCyA-self-dispersible systems, more or less correspond with above-mentioned theory that smaller is better and by this principle bridged liquid based dosage forms with solid dosage forms. Bioavailability of novel drug delivery system which gives coarse dispersion with average particle size between 1-150 microm when dispersed have been tested on healthy volunteers. No difference among pharmacokinetic parameters of novel drug delivery system and microemulsion system have been observed in spite of fact that average particle size of novel system is almost 1000x bigger.     

Preparation and characterization of electrospun PLGA/gelatin nanofibers as a drug delivery system by emulsion electrospinning

Novel biocompatible poly(lactide-co-glycolide) (PLGA) nanofiber mats with favorable biocompatibility and good mechanical strength were prepared, which could serve as an innovative type of tissue engineering scaffold or an ideal controllable drug delivery system. Both hydrophobic and hydrophilic drugs, Cefradine and 5-fluorouracil were successfully loaded into PLGA nanofiber mats by emulsion electrospinning. The natural bioactive protein gelatin (GE) was incorporated into the nanofiber mats to improve the surface properties of the materials for cell adhesion. Nanofibrous scaffolds were characterized by scanning electron microscopy, X-ray diffraction, differential scanning calorimetry, contact angle and tensile measurements. Emulsion electrospun fibers with GE had perfect hydrophilic and good mechanical property. The in vitro release test showed thedrugs released from emulsion electrospun fibers, which achieved lower burst release. The cells cytotoxicity experiment indicated that emulsion electrospun fibers were less toxic and tended to promote fibroblasts cells attachment and proliferation, which implied that the electrospun fibers had promising potential application in tissue engineering or drug delivery.     

Intraocular injection of tamoxifen-loaded nanoparticles: a new treatment of experimental autoimmune uveoretinitis

In this study, we tested the efficiency of an intravitreal injection of tamoxifen, a non-steroidal estrogen receptor modulator, in retinal soluble antigen (S-Ag)-induced experimental autoimmune uveoretinitis (EAU). To increase the bioavailability of tamoxifen, we incorporated tamoxifen into polyethylene glycol (PEG)-coated nanoparticles (NP-PEG-TAM). The localization of the nanoparticles within the eye was investigated using fluorescent-labeled PEG-coated nanoparticles after injection into the vitreous cavity of rats with EAU. Some nanoparticles were distributed extracellularly throughout the ocular tissues, others were concentrated in resident ocular cells and in infiltrating macrophages. Whereas the injection of free tamoxifen did not alter the course of EAU, injection of NP-PEG-TAM performed 1-2 days before the expected onset of the disease in controls resulted in significant inhibition of EAU. NP-PEG-TAM injection significantly reduced EAU compared to injection of NP-PEG-TAM with 17beta-estradiol (E2), suggesting that tamoxifen is acting as a partial antagonist to E2. Diminished infiltration by MHC class II(+) inflammatory cells and low expression of TNF-alpha, IL-1beta, and RANTES mRNA were noted in eyes of NP-PEG-TAM-treated rats. Intravitreal injection of NP-PEG-TAM decreased S-Ag lymphocyte proliferation, IFN-gamma production by inguinal lymph node cells, and specific delayed-type hypersensitivity indicative of a reduced Th1-type response. It increased the anti-S-Ag IgG1 isotype indicating an antibody class switch to Th2 response. These data suggest that NP-PEG-TAM inhibition of EAU could result from a form of immune deviation. Tamoxifen-loaded nanoparticles may represent a new option for the treatment of experimental uveitis.     

载药硼酸盐骨水泥的制备及其体外生物活性和抗菌性能的研究

目的以硼酸盐生物活性玻璃和改性壳聚糖液相制备了新型的硼酸盐骨水泥,同时负载骨髓炎治疗药物硫酸庆大霉素,考察其体外抗菌性能,以探讨其治疗骨髓炎的可能性。方法以硼酸盐骨水泥为载体,制备了负载硫酸庆大霉素(GS)的骨水泥。探究负载GS对骨水泥的可注射性能、初凝时间的影响;将负载GS且预固化的硼酸盐骨水泥浸泡于磷酸盐缓冲溶液(PBS)中,考察其体外生物活性、生物降解性和药物释放;利用抑菌圈实验评估了负载GS的硼酸盐骨水泥的体外抗菌性能。结果制备的载药硼酸盐骨水泥能够被完全注射,初凝时间约6 min;在体外的磷酸盐缓冲溶液(PBS)中浸泡时,GS能够持续稳定的释放,药物释放长达26天;负载GS的硼酸盐骨水泥能够很好地抑制金黄色葡萄球菌(S.aureus)和大肠杆菌(E.coli)的生长。结论制备的负载GS的硼酸盐骨水泥具有优异的可注射性,合适的原位自固化时间,长期持续的药物释放和抗菌性能,可以用于骨髓炎治疗的进一步研究。     

Delivery of membrane impermeable cargo into CHO cells by peptide nanoparticles targeted by a protein corona

Nanocarriers can fulfill essential functions in the stabilization and delivery of drugs: they prevent solubility issues and degradation, reduce side effects and modify the pharmacokinetic profile. However, particle based pharmaceuticals are complex and thus challenging to scale up. As formulation routines account for a large fraction of production costs, reducing complexity in the process of assembly, loading and functionalization of nanoparticles is desirable. Unlike existing approaches with similar goals, our protocol is designed to minimize usage of material and time. Prerequisite to this elegant one-step-procedure is the controlled phase-separation of a hydrophobic peptide to nanoparticles, inducing concurrent cargo-entrapment and association of a protein corona. We demonstrate the process by assembling Flutax-2 containing peptide nanoparticles functionalized with transferrin. Cellular uptake of the particles and cargo release depend on specific particle-cell interactions via transferrin receptor. These data indicate corona-mediated delivery of membrane impermeable cargo in vitro by a particulate delivery system entirely composed of amino acids.     

Alleviation of rheumatoid arthritis by cell-transducible methotrexate upon transcutaneous delivery

Rheumatoid arthritis (RA) is a systemic autoimmune disease that is initiated and maintained by various inflammatory/immune cells and their cytokines, leading to cartilage degradation and bone erosion. Despite its potent therapeutic efficacy on RA, the oral administration of methotrexate (MTX) provokes serious adverse systemic complications, thus necessitating the local application of MTX. Here, we show that transcutaneous MTX (TC-MTX) can efficiently penetrate joint skin ex vivo and in vivo, and that TC-MTX can significantly improve the various inflammatory symptoms associated with RA. Further, TC-MTX preserved the joint-structures in mice with collagen-induced arthritis (CIA), which was also confirmed by three-dimensional micro-computed tomography scan. TC-MTX markedly decreased the secretion of inflammatory cytokines both in the serum and in inflamed joints of CIA mice. Further, its therapeutic potential is comparable to that of etanercept, a biological agent that block tumor necrosis factor (TNF)-α. Importantly, the systemic cytotoxicity of TC-MTX was not detected. Thus, TC-MTX can be a new therapeutic modality for RA patients without systemic complications.     

Nanoparticle-directed sub-cellular localization of doxorubicin and the sensitization breast cancer cells by circumventing GST-Mediated drug resistance

Resistance to single or multiple chemotherapeutic drugs is a major complication in clinical oncology and is one of the most common treatment limitations in patients with reoccurring cancers. Nanoparticle (NP)-based drug delivery systems (DDS's) have been shown to overcome drug resistance in cancer cells mainly by avoiding the activation of efflux pumps in these cells. We demonstrate in this work that polyester-based hyperbranched dendritic-linear (HBDL)-based NPs carrying doxorubicin (Dox) can effectively overcome microsomal glutathione transferase 1 (MGST1)-mediated drug resistance in breast cancer cells. Our DDS was much more effective at considerably lower intracellular Dox concentrations (IC₅₀ 6.3 μm vs. 36.3 μm) and achieved significantly greater reductions in viability and induced higher degrees of apoptosis (31% vs. 14%) compared to the free drug in the resistant cells. Dox-loaded HBDL NPs were found to translocate across the membranes of resistant cells via active endocytic pathways and to be transported to lysosomes, mitochondria, and the endoplasmic reticulum. A significantly lower amount of Dox accumulated in these cytoplasmic compartments in resistant cells treated with free Dox. Moreover, we found that Dox-HBDL significantly decreased the expression of MGST1 and enhanced mitochondria-mediated apoptotic cell death compared to free Dox. Dox-HBDL also markedly activated the JNK pathway that contributes to the apoptosis of drug-resistant cells. These results suggest that HBDL NPs can modulate subcellular drug distribution by specific endocytic and trafficking pathways and that this results in drug delivery that alters enzyme levels and cellular signaling pathways and, most importantly, increases the induction of apoptosis. Our findings suggest that by exploiting the cell transport machinery we can optimize the polymeric vehicles for controlled drug release to overcome drug resistance combat drug resistance with much higher efficacy.     

The control of cargo release from physically crosslinked hydrogels by crosslink dynamics

Controlled release of drugs and other cargo from hydrogels has been an important target for the development of next generation therapies. Despite the increasingly strong focus in this area of research, very little of the published literature has sought to develop a fundamental understanding of the role of molecular parameters in determining the mechanism and rate of cargo release. Herein, a series of physically crosslinked hydrogels have been prepared utilizing host-guest binding interactions of cucurbit[8]uril that are identical in strength (plateau modulus), concentration and structure, yet exhibit varying network dynamics on account of the use of different guests for supramolecular crosslinking. The diffusion of molecular cargo through the hydrogel matrix and the release characteristics from these hydrogels were investigated. It was determined that the release processes of the hydrogels could be directly correlated with the dynamics of the physical interactions responsible for crosslinking and corresponding time-dependent mesh size. These observations highlight that network dynamics play an indispensable role in determining the release mechanism of therapeutic cargo from a hydrogel, identifying that fine-tuning of the release characteristics can be gained through rational design of the molecular processes responsible for crosslinking in the carrier hydrogels.     

Actively targeted delivery of anticancer drug to tumor cells by redox-responsive star-shaped micelles

In cancer therapy nanocargos based on star-shaped polymer exhibit unique features such as better stability, smaller size distribution and higher drug capacity in comparison to linear polymeric micelles. In this study, we developed a multifunctional star-shaped micellar system by combination of active targeting ability and redox-responsive behavior. The star-shaped micelles with good stability were self-assembled from four-arm poly(ε-caprolactone)-poly(ethylene glycol) copolymer. The redox-responsive behaviors of these micelles triggered by glutathione were evaluated from the changes of micellar size, morphology and molecular weight. In vitro drug release profiles exhibited that in a stimulated normal physiological environment, the redox-responsive star-shaped micelles could maintain good stability, whereas in a reducing and acid environment similar with that of tumor cells, the encapsulated agent was promptly released. In vitro cellular uptake and subcellular localization of these micelles were further studied with confocal laser scanning microscopy and flow cytometry against the human cervical cancer cell line HeLa. In vivo and ex vivo DOX fluorescence imaging displayed that these FA-functionalized star-shaped micelles possessed much better specificity to target solid tumor. Both the qualitative and quantitative results of the antitumor effect in 4T1 tumor-bearing BALB/c mice demonstrated that these redox-responsive star-shaped micelles have a high therapeutic efficiency to artificial solid tumor. Therefore, the multifunctional star-shaped micelles are a potential platform for targeted anticancer drug delivery.     

Transdermal immunization with large proteins by means of ultradeformable drug carriers

By means of novel, ultradeformable and self-optimizing agent carriers called transfersomes, large molecules can be brought into the body through intact permeability barriers. This permits non-invasive immunization through normal skin and gives rise to a similar or even slightly higher antibody titer than subcutaneous injections of the same immunogen formulation. The former type of immunization also results in a higher IgA/IgG ratio in the blood than the repeated immunogen injections, as shown here for a soluble protein, human serum albumin, as well as for an integral membrane protein, gap junction protein, in mice.     

Tumor targeting of a cell penetrating peptide by fusing with a pH-sensitive histidine-glutamate co-oligopeptide

Cell penetrating peptides (CPPs) have been well established as potential carriers for intracellular delivery of protein/peptide therapeutics. However, their lack of selectivity impedes their application in vivo. In order to increase their specificity, a highly pH-sensitive histidine-glutamate (HE) co-oligopeptide was fused with a CPP, i.e. model amphipathic peptide (MAP), and was expressed as a fusion protein with glutathione S-transferase (GST) acting as a cargo protein. Compared with two other fusion proteins containing either HE or MAP, only the fused peptide (HE-MAP) could effectively deliver the cargo GST protein to cells at pH 6.5 or below, while maintaining low delivery to cells at pH 7.0 and above. Using a xenograft mouse model of human breast cancer, fluorescent imaging showed that only HE-MAP could effectively target GST to the tumor site, while reducing non-specific association of MAP in other organs. The data presented in this report demonstrate the diagnostic and/or therapeutic potential of the fused peptide, HE-MAP, for targeting the acidic tumor microenvironment. The concise design for this pH-sensitive peptide offers a simple way to overcome CPP's lack of selectivity, which could lead to increased application of CPPs and macromolecular therapeutics.     

Reversion of multidrug resistance by a pH-responsive cyclodextrin-derived nanomedicine in drug resistant cancer cells

Multidrug resistance (MDR) is one of the major problems responsible for inefficiency of cancer chemotherapy. Currently, there is still unmet demand for innovative strategies as well as effective and safe sensitizers to overcome MDR. In this study, we developed a nanosensitizer based on a pH-responsive nanoparticle (NP) derived from acetalated α-cyclodextrin (Ac-aCD). This pH-responsive NP could be effectively endocytosed by MDR cancer cells, and intracellularly transported by endolysosomal compartments. Ac-aCD NP was able to dramatically potentiate the activity of anticancer drugs including paclitaxel, docetaxel, cis-diamminedichloroplatinum, camptothecin, and doxorubicin. This sensitizing capability of Ac-aCD NP on MDR cells was resulted from the combined effects of decreased Pgp expression, attenuated Pgp ATPase activity, and the reduced intracellular ATP level. Ac-aCD NP exerted these diverse biological functions by intracellularly released α-cyclodextrin molecules, which were produced due to hydrolysis of Ac-aCD in acidic subcellular organelle. On the other hand, treatment with Ac-aCD NP showed no significant effects on the integrity of the plasma membrane, cytoskeleton, cell cycle, mitochondrial membrane potential, and apoptosis. These findings suggest that this pH-responsive NP has great potential for effective therapy of resistant cancers by combining with chemotherapeutic agents. It may also serve as a pharmacologically active nanocarrier for intracellular delivery of a plethora of antitumor drugs.     

Endocytosis of PEGylated nanoparticles accompanied by structural and free energy changes of the grafted polyethylene glycol

Nanoparticles (NPs) are in use to efficiently deliver drug molecules into diseased cells. The surfaces of NPs are usually grafted with polyethylene glycol (PEG) polymers, during so-called PEGylation, to improve water solubility, avoid aggregation, and prevent opsonization during blood circulation. The interplay between grafting density σp${\sigma }_{\text{p}}$ and grafted PEG polymerization degree N$N$ makes cellular uptake of PEGylated NPs distinct from that of bare NPs. To understand the role played by grafted PEG polymers, we study the endocytosis of 8 nm sized PEGylated NPs with different σp${\sigma }_{\text{p}}$ and N$N$ through large scale dissipative particle dynamics (DPD) simulations. The free energy change Fpolymer$F_{\text{polymer}}$ of grafted PEG polymers, before and after endocytosis, is identified to have an effect which is comparable to, or even larger than, the bending energy of the membrane during endocytosis. Based on self-consistent field theory Fpolymer$F_{\text{polymer}}$ is found to be dependent on both σp${\sigma }_{\text{p}}$ and N$N$. By incorporating Fpolymer$F_{\text{polymer}}$, the critical ligand-receptor binding strength for PEGylated NPs to be internalized can be correctly predicted by a simple analytical equation. Without considering Fpolymer$F_{\text{polymer}}$, it turns out impossible to predict whether the PEGylated NPs will be delivered into the diseased cells. These simulation results and theoretical analysis not only provide new insights into the endocytosis process of PEGylated NPs, but also shed light on the underlying physical mechanisms, which can be utilized for designing efficient PEGylated NP-based therapeutic carriers with improved cellular targeting and uptake.     

Altering the release of tobramycin by incorporating poly(ethylene glycol) into model silicone hydrogel contact lens materials

Abstract

Delivery of drugs from contact lens materials is attractive for a number of reasons. However, the controlled delivery of hydrophilic drugs can be difficult to achieve due to the burst release of drug that is associated with materials of high water content, such as hydrogels. Silicone hydrogels have significant potential for drug delivery due to their increased hydrophobicity and the tortuous nature of the pores, overcoming some of the limitations associated with conventional hydrogel materials. The aim of this study was to examine the potential of model poly(ethylene glycol) (PEG) containing silicone hydrogels for delivery of hydrophilic aminoglycoside antibiotics. It was hypothesized that PEG, a polymer that has seen extensive use in biomedical applications, will provide in addition to hydrophilicity and protein repulsion, a mechanism for controlling the delivery of this hydrophilic antibiotic. PEG was combined with the macromer TRIS to create the model silicone hydrogel materials. The optical and physical properties of the novel TRIS-co-PEG silicone hydrogels exhibited excellent transparency, appropriate refractive index and high transmittance indicating minimal phase separation. Desirable properties such as wettability and protein repulsion were maintained across a wide range of formulations. The water content was found to be highly correlated with the ethylene oxide content. Drug release could be influenced through PEG content and was found to fit Higuchi-like kinetics. Overall, the study demonstrates that incorporation of PEG into a model silicone hydrogel could be used to control the release of a hydrophilic compound. Data suggests this is related to the unique structure and properties of PEG, which alter the types of water found in each formulation and the water content.     

Controlled dual delivery of BMP-2 and dexamethasone by nanoparticle-embedded electrospun nanofibers for the efficient repair of critical-sized rat calvarial defect

There is an urgent need to develop biomimetic bone tissue engineering scaffolds for the repair of critical-sized calvarial defect. In this study, we developed a new nanoparticle-embedded electrospun nanofiber scaffold for the controlled dual delivery of BMP-2 and dexamethasone (DEX). The scaffold was achieved by (1) the encapsulation of BMP-2 into bovine serum albumin (BSA) nanoparticles to maintain the bioactivity of BMP-2 and (2) the co-electrospinning of the blending solution composed of the BSA nanoparticles, DEX and the poly(ε-caprolactone)-co-poly(ethylene glycol) (PCE) copolymer. The in vitro studies showed that the bioactivity of DEX and BMP-2 was preserved in the dual-drug-loaded nanofiber scaffold, and a sequential release pattern in which most of the DEX was released in the original eight days and the BMP-2 release lasted up to 35 days was achieved. The in vitro osteogenesis study demonstrated that the drug-loaded groups exhibited a strong ability to induce differentiation toward osteoblasts. In vivo osteogenesis studies also revealed that the degrees of repair of rat calvarial defect achieved with the drug-loaded nanofiber scaffolds were significantly better than those obtained with the blank materials; in particular, the dual-drug-loaded nanofiber scaffold manifested the best repair efficacy due to a synergistic effect of BMP-2 and DEX. Therefore, the dual-drug-loaded nanofiber scaffold is deemed a strong potential candidate for the repair of bone defects in bone tissue engineering.     

Finite and infinitesimal representations of the vasculature: ocular drug clearance by vascular and hydraulic effects

New methods are presented for simulating steady-state drug concentration in vitreous, choroid, and sclera from an intravitreal device. Clearance by choroidal flow and intraocular pressure (IOP) -induced Darcy hydraulic flow are included. Two methods are proposed for modeling the vasculature using simple one-dimensional models for simulating drug concentration profiles from intravitreal devices. The finite choroid method adds a concentration-dependent sink term to the convective diffusion equation, allowing for a continuous but rapid decrease in concentration throughout the choroid region. The infinitesimal choroid method uses a combination of a simple flux boundary condition and a redefinition of the dependent variable to account for the impact of the vascular drain by a discontinuous drop in concentration across the exterior vitreous boundary. This eliminates the need for a choroidal region, reducing finite element memory requirements, enabling the choroid to be made arbitrarily thin. The impact of permeating fluid induced by IOP on convection was examined, using hydraulic coefficients for ocular tissue recently made available [Xu, J. [et_al.], Pharm. Res. 17:664–669 (2000)], allowing for drug efflux through the outer sclera. Transport becomes diffusion limited at high vascular clearance. Hydraulic flow restricts the range in concentration predicted in the vitreous compared to zero flow. Hydraulic influences for small, rapidly cleared drugs can be neglected. © 2002 Biomedical Engineering Society. PAC2002: 8719Uv, 4266Ct, 8780-y     

Modulation of muscle contraction by a cell-permeable peptide

In contrast to immortal cell lines, primary cells are hardly susceptible to intracellular delivery methods such as transfection. In this study, we evaluated the direct delivery of several cell-permeable peptides under noninvasive conditions into living primary adult rat cardiomyocytes. We specifically monitored the functional effects of a cell-permeable peptide containing the 15 amino acid N-terminal peptide from human ventricular light chain-1 (VLC-1) on contraction and intracellular Ca2+ signals after electrical stimulation in primary adult cardiomyocytes. The transducible VLC-1 variant was taken up by cardiomyocytes within 5 min with more than 95% efficiency and localized to sarcomeric structures. Analysis of the functional effects of the cell-permeable VLC-1 revealed an enhancement of the intrinsic contractility of cardiomyocytes without affecting the intracellular Ca2+. Therefore, peptide transduction mediated by cell-penetrating peptides represents not only a unique strategy to enhance heart muscle function with no secondary effect on intracellular Ca2+ but also an invaluable tool for the modulation and manipulation of protein interactions in general and in primary cells.