Thermoresponsive- co-Biodegradable Linear-Dendritic Nanoparticles for Sustained Release of Nerve Growth Factor To Promote Neurite Outgrowth.

Thermoresponsive and biodegradable linear-dendritic nanoparticles containing poly( N-isopropylacrylamide), poly(l-lactic acid), and poly(l-lysine) dendrons were investigated for sustained release of nerve growth factor (NGF) in response to temperature change. The nanoparticles and their degradants were not cytotoxic to neuron-like PC12 cells for at least one month. The nanoparticles were preferentially taken up by PC12 cells 6-13-times more at temperatures above (37 °C) than below (25 °C) the lower critical solution temperature of the nanoparticles. NGF could be loaded into the nanoparticles in aqueous solution and slowly released from the nanoparticles for 12 and 33 days at 25 and 37 °C, respectively. The released NGF was biologically active by promoting neurite outgrowth of PC12 cells. This work demonstrates a new concept of using thermoresponsive and biodegradable linear-dendritic nanoparticles for thermally targeted and sustained release of NGF and other protein drugs for the treatment of Alzheimer's disease and other neurological disorders.

Long-acting injectable hormonal dosage forms for contraception.

Although great efforts have been made to develop long-acting injectable hormonal contraceptives for more than four decades, few long-acting injectable contraceptives have reached the pharmaceutical market or even entered clinical trials. On the other hand, in clinical practice there is an urgent need for injectable long-acting reversible contraceptives which can provide contraceptive protection for more than 3 months after one single injection. Availability of such products will offer great flexibility to women and resolve certain continuation issues currently occurring in clinics. Herein, we reviewed the strategies exploited in the past to develop injectable hormonal contraceptive dosages including drug microcrystal suspensions, drug-loaded microsphere suspensions and in situ forming depot systems for long-term contraception and discussed the potential solutions for remaining issues met in the previous development.

Overcoming the blood-brain barrier in chemotherapy treatment of pediatric brain tumors.

Pediatric brain tumors are most common cancers in childhood and among the leading causes of death in children. Chemotherapy has been used as adjuvant (i.e. after) or neoadjuvant (i.e. before) therapy to surgery and radiotherapy for the management of pediatric brain tumors for more than four decades and gained more attention in the recent two decades. Although chemotherapy has demonstrated its effectiveness in the management of some pediatric brain tumors, failure or inactiveness of chemotherapy is commonly met in the clinics and clinical trials. Some of these failures might be attributed to the blood-brain barrier (BBB), limiting the penetration of systemically administered chemotherapeutics into pediatric brain tumors. Therefore, various strategies have been developed and used to address this issue. Herein, we review different methods reported in the literature to circumvent the BBB for enhancing the present of chemotherapeutics in the brain to treat pediatric brain tumors.

Designing Biomimetic 3D-Printed Osteochondral Scaffolds for Enhanced Load-Bearing Capacity.

Osteoarthritis is a debilitating chronic joint disorder that affects millions of people worldwide. Since palliative and surgical treatments cannot completely regenerate hyaline cartilage within the articulating joint, osteochondral (OC) tissue engineering has been explored to heal OC defects. Utilizing computational simulations and three-dimensional (3D) printing, we aimed to build rationale around fabricating OC scaffolds with enhanced biomechanics. First, computational simulations revealed that interfacial fibrils within a bilayer alter OC scaffold deformation patterns by redirecting load-induced stresses toward the top of the cartilage layer. Principal component analysis revealed that scaffolds with 800 µm long fibrils (scaffolds 8A-8H) possessed optimal biomechanical properties to withstand compression and shear forces. While compression testing indicated that OC scaffolds with 800 µm fibrils did not have greater compressive moduli than other scaffolds, interfacial shear tests indicated that scaffold 8H possessed the greatest shear strength. Lastly, failure analysis demonstrated that yielding or buckling models describe interfacial fibril failure depending on fibril slenderness S. Specifically for scaffolds with packing density n = 6 and n = 8, the yielding failure model fits experimental loads with S < 10, while the buckling model fitted scaffolds with S < 10 slenderness. The research presented provides critical insights into designing 3D printed interfacial scaffolds with refined biomechanics toward improving OC tissue engineering outcomes.

DeepFreeze 3D-biofabrication for Bioengineering and Storage of Stem Cells in Thick and Large-Scale Human Tissue Analogs.

3D bioprinting holds great promise for meeting the increasing need for transplantable tissues and organs. However, slow printing, interlayer mixing, and the extended exposure of cells to non-physiological conditions in thick structures still hinder clinical applications. Here the DeepFreeze-3D (DF-3D) procedure and bioink for creating multilayered human-scale tissue mimetics is presented for the first time. The bioink is tailored to support stem cell viability, throughout the rapid freeform DF-3D biofabrication process. While the printer nozzle is warmed to room temperature, each layer solidifies at contact with the stage (-80 °C), or the subsequent layers, ensuring precise separation. After thawing, the encapsulated stem cells remain viable without interlayer mixing or delamination. The composed cell-laden constructs can be cryogenically stored and thawed when needed. Moreover, it is shown that under inductive conditions the stem cells differentiate into bone-like cells and grow for months after thawing, to form large tissue-mimetics in the scale of centimeters. This is important, as this approach allows the generation and storage of tissue mimetics in the size and thickness of human tissues. Therefore, DF-3D biofabrication opens new avenues for generating off-the-shelf human tissue analogs. It further holds the potential for regenerative treatments and for studying tissue pathologies caused by disease, tumor, or trauma.

Injectable systems for long-lasting insulin therapy.

Insulin therapy is the mainstay to treat diabetes characterizedd by hyperglycemia. However, its short half-life of only 4-6 min limits its effectiveness in treating chronic diabetes. Advances in recombinant DNA technology and protein engineering have led to several insulin analogue products that have up to 42 h of glycemic control. However, these insulin analogues still require once- or twice-daily injections for optimal glycemic control and have poor patient compliance and adherence issues. To achieve insulin release for more than one day, different injectable delivery systems including microspheres, in situ forming depots, nanoparticles and composite systems have been developed. Several of these delivery systems have advanced to clinical trials for once-weekly insulin injection. This review comprehensively summarizes the developments of injectable insulin analogs and delivery systems covering the whole field of injectable long-lasting insulin technologies from prototype design, preclinical studies, clinical trials to marketed products for the treatment of diabetes.

ß-cyclodextrin-poly(ß-amino ester) nanoparticles for sustained drug delivery across the blood-brain barrier.

Novel biodegradable polymeric nanoparticles composed of ß-cyclodextrin and poly(ß-amino ester) segments have been developed for sustained drug delivery across the blood-brain barrier (BBB). The nanoparticles have been synthesized by cross-linking ß-cyclodextrin with poly(ß-amino ester) via the Michael addition method. The chemical, physical, and degradation properties of the nanoparticles have been characterized by matrix-assisted laser desoption/ionization time-of-flight, attenuated total reflectance Fourier transform infrared spectroscopy, nuclear magnetic resonance, dynamic light scattering, and atomic force microscopy techniques. Bovine and human brain microvascular endothelial cell monolayers have been constructed as in vitro BBB models. Preliminary results show that the nanoparticles do not affect the integrity of the in vitro BBB models, and the nanoparticles have much higher permeability than dextran control across the in vitro BBB models. Doxorubicin has been loaded into the nanoparticles with a loading efficiency of 86%, and can be released from the nanoparticles for at least one month. The developed ß-cyclodextrin-poly(ß-amino ester) nanoparticles might be useful as drug carriers for transporting drugs across the BBB to treat chronic diseases in the brain.

Design strategies for programmable oligonucleotide nanotherapeutics.

A systematic review on how to design different programmable nanotherapeutics using oligonucleotides as building blocks or as surface and matrix modifiers for controlled and targeted delivery of various therapeutic agents in presented.

Quaternary ammonium beta-cyclodextrin nanoparticles for enhancing doxorubicin permeability across the in vitro blood-brain barrier.

This study describes novel quaternary ammonium beta-cyclodextrin (QAbetaCD) nanoparticles as drug delivery carriers for doxorubicin (DOX), a hydrophobic anticancer drug, across the blood-brain barrier (BBB). QAbetaCD nanoparticles show 65-88 nm hydrodynamic radii with controllable cationic properties by adjusting the incorporated amount of quaternary ammonium group in their structure. ATR-FTIR studies confirm the complexation between the QAbetaCD nanoparticles and DOX. QAbetaCD nanoparticles are not toxic to bovine brain microvessel endothelial cells (BBMVECs) at concentrations up to 500 microg x mL(-1). They also do not change the integrity of BBMVEC monolayers, an in vitro BBB model, including transendothelial electrical resistance value, Lucifer yellow permeability, tight junction protein occludin and ZO-1 expression and morphology, cholesterol extraction, and P-glycoprotein (P-gp) expression and efflux activity, at a concentration of 100 microg x mL(-1). Some QAbetaCD nanoparticles not only are twice as permeable as dextran (M(w) = 4000 g x mol(-1)) control, but also enhance DOX permeability across BBMVEC monolayers by 2.2 times. Confocal microscopy and flow cytometry measurements imply that the permeability of QAbetaCD nanoparticles across the in vitro BBB is probably due to endocytosis. DOX/QAbetaCD complexes kill U87 cells as effectively as DOX alone. However, QAbetaCD nanoparticles completely protect BBMVECs from cytotoxicity of DOX at 5 and 10 microM after 4 h incubation. The developed QAbetaCD nanoparticles have great potential in safely and effectively delivering DOX and other therapeutic agents across the BBB.

Design strategies for chemical-stimuli-responsive programmable nanotherapeutics.

Chemical-stimuli-responsive nanotherapeutics have gained great interest in drug delivery and diagnosis applications. These nanotherapeutics are designed to respond to specific internal stimuli including pH, ionic strength, redox, reactive oxygen species, glucose, enzymes, ATP and hypoxia for site-specific and responsive or triggered release of payloads and/or biomarker detections. This review systematically and comprehensively addresses up-to-date technological and design strategies, and challenges nanomaterials to be used for triggered release and sensing in response to chemical stimuli.

Nanotechnology in regenerative ophthalmology.

In recent years, regenerative medicine is gaining momentum and is giving hopes for restoring function of diseased, damaged, and aged tissues and organs and nanotechnology is serving as a catalyst. In the ophthalmology field, various types of allogenic and autologous stem cells have been investigated to treat some ocular diseases due to age-related macular degeneration, glaucoma, retinitis pigmentosa, diabetic retinopathy, and corneal and lens traumas. Nanomaterials have been utilized directly as nanoscaffolds for these stem cells to promote their adhesion, proliferation and differentiation or indirectly as vectors for various genes, tissue growth factors, cytokines and immunosuppressants to facilitate cell reprogramming or ocular tissue regeneration. In this review, we reviewed various nanomaterials used for retina, cornea, and lens regenerations, and discussed the current status and future perspectives of nanotechnology in tracking cells in the eye and personalized regenerative ophthalmology. The purpose of this review is to provide comprehensive and timely insights on the emerging field of nanotechnology for ocular tissue engineering and regeneration.

Thermoresponsive and biodegradable linear-dendritic nanoparticles for targeted and sustained release of a pro-apoptotic drug.

Ceramide is a bioactive sphingolipid-derived second messenger that has been demonstrated to induce apoptosis and cell cycle arrest in various cancer cell culture systems. Although in vitro tumor cell culture models have illuminated the potential therapeutic utility of a cell-permeable analog of ceramide, C(6), in vivo delivery is impeded by the extreme hydrophobicity and physical-chemical properties of this bioactive lipid. Previously, we have demonstrated that the incorporation of C(6) into pegylated liposomal vesicles is an effective anti-cancer drug delivery strategy in vitro and in vivo. Here, we report the utilization of a novel multi-functional polymeric drug delivery system designed to therapeutically target C(6) to solid tumor tissue. This delivery system is a hydrolytically degradable and temperature-sensitive linear-dendritic nanoparticle with a lower critical solution temperature (LCST) of 30 degrees C. C(6) was effectively loaded into the nanoparticles, and released continuously for at least 1 month in vitro, measured by mass spectroscopy. The preferential uptake of fluorescein isothiocyanate-labeled linear-dendritic nanoparticles into human MDA-MB-231 breast adenocarcinoma cells at temperature above the LCST (37 degrees C) was confirmed by confocal microscopy and quantified by flow cytometry. The accumulation of NBD-C(6) into MDA-MB-231 cells was highly enhanced by the thermoresponsive linear-dendritic nanoparticles, but not by non-thermoresponsive liposome and PEG-dendritic polymer, at temperature above the LCST (37 degrees C). The linear-dendritic nanoparticles alone were not toxic, but their complexes with C(6) caused significant growth inhibition and apoptosis to MDA-MB-231 cells at 37 degrees C. The designed thermoresponsive and biodegradable linear-dendritic nanoparticles have great potential for thermally targeted and sustained release of C(6) for the treatment of solid tumors with hyperthermia.

Design strategies for physical-stimuli-responsive programmable nanotherapeutics.

Nanomaterials that respond to externally applied physical stimuli such as temperature, light, ultrasound, magnetic field and electric field have shown great potential for controlled and targeted delivery of therapeutic agents. However, the body of literature on programming these stimuli-responsive nanomaterials to attain the desired level of pharmacologic responses is still fragmented and has not been systematically reviewed. The purpose of this review is to summarize and synthesize the literature on various design strategies for simple and sophisticated programmable physical-stimuli-responsive nanotherapeutics.

Porous thermoresponsive-co-biodegradable hydrogels as tissue-engineering scaffolds for 3-dimensional in vitro culture of chondrocytes.

A new porous, thermoresponsive, partially biodegradable, chemically crosslinked hydrogel system was developed, characterized, and tested as a cartilage tissue-engineering scaffold for in vitro chondrocyte culture over a 4-week period. The hydrogel system was composed of poly(N-isopropylacrylamide), poly(D,L-lactic acid), and dextran segments. Pores in the hydrogels were generated using a salt leaching technique. The hydrogels showed thermoresponsive properties, with a lower critical solution temperature at approximately 32 degrees C. They continuously swelled at physiological temperature in phosphate buffered saline (pH 7.4) for at least 1 month. Chondrocytes isolated from embryonic chick sterna were seeded into the hydrogel scaffolds at room temperature and cultured at 37 degrees C for 4 weeks. Real-time reverse-transcriptase polymerase chain reaction quantification was conducted every week to study messenger ribonucleic acid levels of 3 chondrocyte phenotypic markers: type II collagen, type X collagen, and Indian hedgehog. Results suggested that chondrocytes maintained their phenotype during the 4-week in vitro culture and could mimic in vivo development. Chondrocytes were non-enzymatically harvested from the hydrogel scaffold at the end of the fourth week by simply lowering the temperature from 37 degrees C to room temperature. The harvested chondrocytes kept a round morphology, confirming the maintenance of the chondrocyte phenotype in the hydrogel scaffolds.

Nanoparticles for drug delivery to the anterior segment of the eye.

Commercially available ocular drug delivery systems are effective but less efficacious to manage diseases/disorders of the anterior segment of the eye. Recent advances in nanotechnology and molecular biology offer a great opportunity for efficacious ocular drug delivery for the treatments of anterior segment diseases/disorders. Nanoparticles have been designed for preparing eye drops or injectable solutions to surmount ocular obstacles faced after administration. Better drug pharmacokinetics, pharmacodynamics, non-specific toxicity, immunogenicity, and biorecognition can be achieved to improve drug efficacy when drugs are loaded in the nanoparticles. Despite the fact that a number of review articles have been published at various points in the past regarding nanoparticles for drug delivery, there is not a review yet focusing on the development of nanoparticles for ocular drug delivery to the anterior segment of the eye. This review fills in the gap and summarizes the development of nanoparticles as drug carriers for improving the penetration and bioavailability of drugs to the anterior segment of the eye.

Injectable In Situ Forming Depot Systems for Long-Acting Contraception.

Up to date, no long-acting reversible contraceptive (LARC) is developed to be injectable through needles smaller than 18 G and can also provide contraception for more than 3 months after single injection. In this study, injectable polymeric in situ forming depot (ISD) systems are developed to have injectability through 21-23 G needles, and capability of sustained release of levonorgestrel (LNG) for at least 7 months in vitro and in vivo after single subcutaneous injection in rats. The systems are polymeric solutions composed of biodegradable poly(lactide-co-glycolide) and poly(lactic acid) polymers dissolved in a mixture of solvents like N-methyl-2-pyrrolidone and benzyl benzoate or triethyl citrate. LNG released from ISD systems successfully suppressed the estrous cycle of rats at plasma concentration above 0.35 ng mL-1 . At the end of the treatment, when LNG plasma concentration drops down to be nondetectable, predictable return of fertility is observed in rats. The designed ISD systems have great potential to be further developed into robust injectable LARCs that can be injected through a 21 G or smaller needle and achieve a variety of contraception durations with high patient compliance and low cost.

An Accelerated Release Study to Evaluate Long-Acting Contraceptive Levonorgestrel-Containing in Situ Forming Depot Systems.

Biodegradable polymer-based injectable in situ forming depot (ISD) systems that solidify in the body to form a solid or semisolid reservoir are becoming increasingly attractive as an injectable dosage form for sustained (months to years) parenteral drug delivery. Evaluation of long-term drug release from the ISD systems during the formulation development is laborious and costly. An accelerated release method that can effectively correlate the months to years of long-term release in a short time such as days or weeks is economically needed. However, no such accelerated ISD system release method has been reported in the literature to date. The objective of the current study was to develop a short-term accelerated in vitro release method for contraceptive levonorgestrel (LNG)-containing ISD systems to screen formulations for more than 3-month contraception after a single subcutaneous injection. The LNG-containing ISD formulations were prepared by using biodegradable poly(lactide-co-glycolide) and polylactic acid polymer and solvent mixtures containing N-methyl-2-pyrrolidone and benzyl benzoate or triethyl citrate. Drug release studies were performed under real-time (long-term) conditions (PBS, pH 7.4, 37 °C) and four accelerated (short-term) conditions: (A) PBS, pH 7.4, 50 °C; (B) 25% ethanol in PBS, pH 7.4, 50 °C; (C) 25% ethanol in PBS, 2% Tween 20, pH 7.4, 50 °C; and (D) 25% ethanol in PBS, 2% Tween 20, pH 9, 50 °C. The LNG release profile, including the release mechanism under the accelerated condition D within two weeks, correlated (r² ≥ 0.98) well with that under real-time conditions at four months.

Thermoresponsive terpolymeric films applicable for osteoblastic cell growth and noninvasive cell sheet harvesting.

A novel multifunctional linear block copolymer, poly(N-isopropylacrylamide-co-acrylic acid)-b-poly( L-lactic acid) (NAL), was synthesized to expand the concept of cell sheet engineering by using its thermoresponsive property and processibility. The chemical structure of synthesized NAL was confirmed by Fourier transform infrared spectroscopy, and its molar mass (103,500 g.mol(-1)) and molar mass distribution were determined by matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. NAL copolymer was fabricated into thin films by spin-casting. Spin-cast NAL films displayed thermoresponsive properties as demonstrated by surface wettability and topology changes from relatively more hydrophobic (contact angle of 56 degrees) and rougher at 37 degrees C to relatively more hydrophilic (contact angle of 40 degrees) and smoother at 22 degrees C, as assessed by contact angle measurement and atomic force microscopy, respectively. Murine osteoblastic MC3T3-E1 cells displayed comparable adhesion but slower proliferation on NAL films than on poly(L-lactic acid) (PLLA) films and tissue culture polystyrene (TCPS). Within 9 days of cell culture, the highest alkaline phosphatase activity of MC3T3-E1 cells occurred later (on day 9) on NAL films than on PLLA films and TCPS (on day 6). A well-established MC3T3-E1 cell sheet was successfully detached from NAL films, in the absence of enzymes, within about 5 min by simply lowering the temperature from 37 degrees C to room temperature. NAL copolymer has potential for use in the controlled release of therapeutic agents while simultaneously supporting cell growth. In addition, it may be applicable for noninvasive two- or three-dimensional cell sheet harvesting.

Subconjunctivally Implanted Hydrogels for Sustained Insulin Release to Reduce Retinal Cell Apoptosis in Diabetic Rats.

PURPOSE: Diabetic retinopathy (DR) is a leading cause of blindness in diabetic patients that involves early-onset retinal cell loss. Here, we report our recent work using subconjunctivally implantable hydrogels for sustained insulin release to the retina to prevent retinal degeneration. METHODS: The hydrogels are synthesized by UV photopolymerization of N-isopropylacrylamide and a dextran macromer containing oligolactate-(2-hydroxyetheyl methacrylate) units. Insulin was loaded into the hydrogels during the synthesis. The ex vivo bioactivity of insulin released from the hydrogels was tested on fresh rat retinas using immunoprecipitation and immunoblotting to measure insulin receptor tyrosine and Akt phosphorylation. The biosafety and the effect on the blood glucose of the hydrogels were evaluated in rats 2 months after subconjunctival implantation. The release of insulin from the hydrogels was studied both in vitro in PBS (pH 7.4), and in vivo using confocal microscopy and RIA kit. The in vivo bioactivity of the released insulin was investigated in diabetic rats using DNA fragmentation method. RESULTS: The hydrogels could load insulin with approximately 98% encapsulation efficiency and continuously release FITC-insulin in PBS (pH = 7.4) at 37°C for at least 5 months depending on their composition. Insulin lispro released from the hydrogels was biologically active by increasing insulin receptor tyrosine and Akt serine phosphorylation of ex vivo retinas. In vivo studies showed normal retinal histology 2 months post subconjunctival implantation. Insulin released from subconjunctivally implanted hydrogels could be detected in the retina by using confocal microscopy and RIA kit for 1 week. The implanted hydrogels with insulin lispro did not change the blood glucose level of normal and diabetic rats, but significantly reduced the DNA fragmentation of diabetic retinas for 1 week. CONCLUSIONS: The developed hydrogels have great potential to sustain release of insulin to the retina via subconjunctival implantation to minimize DR without the risk of hypoglycemia.

Development and validation of sensitive LC/MS/MS method for quantitative bioanalysis of levonorgestrel in rat plasma and application to pharmacokinetics study.

Rapid, sensitive, selective and accurate LC/MS/MS method was developed for quantitative determination of levonorgestrel (LNG) in rat plasma and further validated for specificity, linearity, accuracy, precision, sensitivity, matrix effect, recovery efficiency and stability. Liquid-liquid extraction procedure using hexane:ethyl acetate mixture at 80:20 v:v ratio was employed to efficiently extract LNG from rat plasma. Reversed phase Luna column C18(2) (50×2.0mm i.d., 3µM) installed on a AB SCIEX Triple Quad™ 4500 LC/MS/MS system was used to perform chromatographic separation. LNG was identified within 2min with high specificity. Linear calibration curve was drawn within 0.5-50ng·mL(-1) concentration range. The developed method was validated for intra-day and inter-day accuracy and precision whose values fell in the acceptable limits. Matrix effect was found to be minimal. Recovery efficiency at three quality control (QC) concentrations 0.5 (low), 5 (medium) and 50 (high) ng·mL(-1) was found to be >90%. Stability of LNG at various stages of experiment including storage, extraction and analysis was evaluated using QC samples, and the results showed that LNG was stable at all the conditions. This validated method was successfully used to study the pharmacokinetics of LNG in rats after SubQ injection, providing its applicability in relevant preclinical studies.