Apatite-Polymer Composite Particles for Controlled Delivery of BMP-2: In Vitro Release and Cellular Response

Bone morphogenetic protein-2 (BMP-2) has the ability to induce osteoblast differentiation of undifferentiated cells, resulting in the healing of skeletal defects when delivered with a suitable carrier. We have applied a versatile delivery platform comprising a novel composite of two biomaterials with proven track records – apatite and poly(lactic-co-glycolic acid) (PLGA) – to the delivery of BMP-2. Sustained release of this growth factor was tuned with variables that affect polymer degradation and/or apatite dissolution, such as polymer molecular weight, polymer composition, apatite loading, and apatite particle size. The effect of released BMP-2 on C3H10T1/2 murine pluripotent mesenchymal cells was assessed by tracking the expression of osteoblastic makers, alkaline phosphatase (ALP) and osteocalcin. Release media collected over 100 days induced elevated ALP activity in C3H10T1/2 cells. The expression of osteocalcin was also upregulated significantly. These results demonstrated the potential of apatite-PLGA composite particles for releasing protein in bioactive form over extended periods of time.Singapore-MIT Alliance (SMA

Apatite-Polymer Composite Particles for Controlled Delivery of BMP-2

We have developed a versatile delivery platform comprising a novel composite of two biomaterials with proven track records: apatite and poly(lactic-co-glycolic acid) (PLGA). These composites have been tested in the delivery of a model protein, bovine serum albumin (BSA), as well as a growth factor, bone morphogenetic protein-2 (BMP-2), which is a potent inducer of bone formation. The controlled release strategy is based on the use of a polymer with acidic degradation products to control the dissolution of a basic inorganic component, resulting in protein release. The release profile can be modified systematically by changing variables that affect polymer degradation and/or apatite dissolution, such as polymer molecular weight, polymer composition, apatite loading, and apatite particle size. We have found that an increase in polymer molecular weight and polymer hydrophobicity led to slower polymer degradation, and in turn, slower apatite dissolution and protein release. Protein release was enhanced by reducing apatite particle size and by lowering the apatite content in the composites. We anticipate that this delivery platform can be extended to the controlled release of other therapeutic proteins and chemicals.Singapore-MIT Alliance (SMA

Targeted Stimuli-Responsive Dextran Conjugates for Doxorubicin Delivery to Hepatocytes

A targeted, stimuli-responsive, polymeric drug delivery vehicle is being developed in our lab to help alleviate severe side-effects caused by narrow therapeutic window drugs. Targeting specific cell types or organs via proteins, specifically, lectin-mediated targeting holds potential due to the high specificity and affinity of receptor-ligand interactions, rapid internalization, and relative ease of processing. Dextran, a commercially available, biodegradable polymer has been conjugated to doxorubicin and galactosamine to target hepatocytes in a three-step, one-pot synthesis. The loading of doxorubicin and galactose on the conjugates was determined by absorbance at 485 nm and elemental analysis, respectively. Conjugation efficiency based on the amount loaded of each reactant varies from 20% to 50% for doxorubicin and from 2% to 20% for galactosamine. Doxorubicin has also been attached to dextran through an acid-labile hydrazide bond. Doxorubicin acts by intercalating with DNA in the nuclei of cells. The fluorescence of doxorubicin is quenched when it binds to DNA. This allows a fluorescence-based cell-free assay to evaluate the efficacy of the polymer conjugates where we measure the fluorescence of doxorubicin and the conjugates in increasing concentrations of calf thymus DNA. Fluorescence quenching indicates that our conjugates can bind to DNA. The degree of binding increases with polymer molecular weight and substitution of doxorubicin. In cell culture experiments with hepatocytes, the relative uptake of polymer conjugates was evaluated using flow cytometry, and the killing efficiency was determined using the MTT cell proliferation assay. We have found that conjugate uptake is much lower than that of free doxorubicin. Lower uptake of conjugates may increase the maximum dose of drug tolerated by the body. Also, non-galactosylated conjugate uptake is lower than that of the galactosylated conjugate. Microscopy indicates that doxorubicin localizes almost exclusively at the nucleus, whereas the conjugates are present throughout the cell. Doxorubicin linked to dextran through a hydrazide bond was used to achieve improved killing efficiency. Following uptake, the doxorubicin dissociates from the polymer in an endosomal compartment and diffuses to the nucleus. The LC₅₀ of covalently linked doxorubicin is 7.4 μg/mL, whereas that of hydrazide linked doxorubicin is 4.4 μg/mL.Singapore-MIT Alliance (SMA

Apatite-Polymer Composites for the Controlled Dual Delivery of BMP-2 and BMP-6 for Bone Tissue Engineering

The release of growth factors from tissue engineering scaffolds provides signals that influence the migration, differentiation, and proliferation of cells. The incorporation of a drug delivery platform that is capable of tunable release will give tissue engineers greater versatility in the direction of tissue regeneration. We have prepared a novel composite of two biomaterials with proven track records - apatite and poly(lactic-co-glycolic acid) (PLGA) – as a drug delivery platform with promising controlled release properties. These composites have been tested in the delivery of a model protein, bovine serum albumin (BSA), as well as therapeutic proteins, recombinant human bone morphogenetic protein-2 (rhBMP-2) and rhBMP-6. The controlled release strategy is based on the use of a polymer with acidic degradation products to control the dissolution of the basic apatitic component, resulting in protein release. Therefore, any parameter that affects either polymer degradation or apatite dissolution can be used to control protein release. We have modified the protein release profile systematically by varying the polymer molecular weight, polymer hydrophobicity, apatite loading, apatite particle size, and other material and processing parameters. Biologically active rhBMP-2 was released from these composite microparticles over 100 days, in contrast to conventional collagen sponge carriers, which were depleted in approximately 2 weeks. The released rhBMP-2 was able to induce elevated alkaline phosphatase and osteocalcin expression in pluripotent murine embryonic fibroblasts. To augment tissue engineering scaffolds with tunable and sustained protein release capabilities, these composite microparticles can be dispersed in the scaffolds in different combinations to obtain a superposition of the release profiles. We have loaded rhBMP-2 into composite microparticles with a fast release profile, and rhBMP-6 into slow-releasing composite microparticles. An equi-mixture of these two sets of composite particles was then injected into a collagen sponge, allowing for dual release of the proteins from the collagenous scaffold. The ability of these BMP-loaded scaffolds to induce osteoblastic differentiation in vitro and ectopic bone formation in a rat model is being investigated. We anticipate that these apatite-polymer composite microparticles can be extended to the delivery of other signalling molecules, and can be incorporated into other types of tissue engineering scaffolds.Singapore-MIT Alliance (SMA

Experimental analysis of gene assembly with TopDown one-step real-time gene synthesis

Herein we present a simple, cost-effective TopDown (TD) gene synthesis method that eliminates the interference between the polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. The method involves two key steps: (i) design of outer primers and assembly oligonucleotide set with a melting temperature difference of >10°C and (ii) utilization of annealing temperatures to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. In addition, we have combined the proposed method with real-time PCR to analyze the step-wise efficiency and the kinetics of the gene synthesis process. Gel electrophoresis results are compared with real-time fluorescence signals to investigate the effects of oligonucleotide concentration, outer primer concentration, stringency of annealing temperature, and number of PCR cycles. Analysis of the experimental results has led to insights into the gene synthesis process. We further discuss the conditions for preventing the formation of spurious DNA products. The TD real-time gene synthesis method provides a simple and efficient method for assembling fairly long DNA sequence, and aids in optimizing gene synthesis conditions. To our knowledge, this is the first report that utilizes real-time PCR for gene synthesis

Targeted Stimuli-Responsive Dextran Conjugates for Doxorubicin Delivery to Hepatocytes

A targeted, stimuli-responsive, polymeric drug delivery vehicle has been developed to help alleviate the severe side-effects caused by narrow therapeutic window drugs. Doxorubicin, a commonly used chemotherapeutic agent has been conjugated to dextran by two different techniques. In the first method, doxorubicin and hepatocyte-targeting galactosamine were attached to dextran through amine bonds. Conjugation efficiency based on the amount loaded of each reactant varied from 1% to 50% for doxorubicin and from 2% to 20% for galactosamine, depending on various synthesis parameters. For the second conjugate, doxorubicin was attached to dextran through an acid-labile hydrazide bond. Fluorescence quenching indicated that all our conjugates can bind to DNA. The degree of binding was improved with increasing polymer molecular weight and substitution of doxorubicin, and also with hydrazide-bonded conjugate. In cell culture experiments, we have found that the uptake of conjugates was much lower than that of free doxorubicin. Lower uptake of conjugates decreased the toxicity of doxorubicin. Also, the uptake of non-galactosylated conjugate was lower than that of the galactosylated conjugate. Microscopy studies indicated that doxorubicin was localized almost exclusively at the nucleus, whereas the amine-bonded conjugates were present throughout the cell. Targeted amine-linked conjugates and hydrazide-bonded conjugates achieved greatly improved cytotoxicity. Following uptake, the doxorubicin was dissociated from the hydrazide conjugate in an endosomal compartment and diffused to the nucleus. The LC₅₀ values of non-targeted amine-linked, targeted amine-linked, and hydrazide-linked doxorubicin were 19.81 μg/mL, 7.33 μg/mL and 4.39 μg/mL, respectively. The amine-linked conjugates were also tested on a multidrug-resistant cell line; the LC₅₀ values of doxorubicin and the non-targeted amine-linked conjugate were 8.60 μg/mL and 36.02 μg/mL, respectively.Singapore-MIT Alliance (SMA

Three-photon absorption in water-soluble ZnS nanocrystals

We report on large three-photon absorption (3PA) in glutathione-capped ZnS semiconductor nanocrystals (NCs), determined by both Z-scan and transient transmission techniques with 120-fs laser pulses. The monodispersed, water-soluble ZnS NCs are synthesized by a modified protocol with a mean diameter of 2.5 nm. Their 3PA cross-section is determined to be around 2.7x10^-78 cm^6s^2photon^-2 at an optimal wavelength of commercial Ti:sapphire femtosecond lasers. This value is nearly one order of magnitude greater than that of CdS NCs, and four to five orders of magnitude higher than those of the previously reported common UV fluorescent dyes.Comment: 15 pages, 4 figure

Achievements and challenges in bioartificial kidney development

Bioartificial kidneys (BAKs) combine a conventional hemofilter in series with a bioreactor unit containing renal epithelial cells. The epithelial cells derived from the renal tubule should provide transport, metabolic, endocrinologic and immunomodulatory functions. Currently, primary human renal proximal tubule cells are most relevant for clinical applications. However, the use of human primary cells is associated with many obstacles, and the development of alternatives and an unlimited cell source is one of the most urgent challenges. BAKs have been applied in Phase I/II and Phase II clinical trials for the treatment of critically ill patients with acute renal failure. Significant effects on cytokine concentrations and long-term survival were observed. A subsequent Phase IIb clinical trial was discontinued after an interim analysis, and these results showed that further intense research on BAK-based therapies for acute renal failure was required. Development of BAK-based therapies for the treatment of patients suffering from end-stage renal disease is even more challenging, and related problems and research approaches are discussed herein, along with the development of mobile, portable, wearable and implantable devices

TmPrime: fast, flexible oligonucleotide design software for gene synthesis

Herein we present TmPrime, a computer program to design oligonucleotide sets for gene assembly by both ligase chain reaction (LCR) and polymerase chain reaction (PCR). TmPrime offers much flexibility with no constraints on the gene and oligonucleotide lengths. The program divides the long input DNA sequence based on the input desired melting temperature, and dynamically optimizes the length of oligonucleotides to achieve homologous melting temperatures. The output reports the melting temperatures, oligonucleotide sequences and potential formation of secondary structures. Our program also provides functions on sequence pooling to separate long genes into smaller pieces for multi-pool assembly and codon optimization for expression. The software has been successfully used in the design and synthesis of green fluorescent protein fragment (GFPuv) (760 bp), human protein kinase B-2 (PKB2) (1446 bp) and the promoter of human calcium-binding protein A4 (S100A4) (752 bp) using real-time PCR assembly with LCGreen I, which offers a novel approach to compare the efficiency of gene synthesis. The purity of assembled products is successfully estimated with the use of melting curve analysis, which would potentially eliminate the necessity for agarose gel electrophoresis. This program is freely available at http://prime.ibn.a-star.edu.sg

World Health Organization cardiovascular disease risk charts: revised models to estimate risk in 21 global regions

BACKGROUND: To help adapt cardiovascular disease risk prediction approaches to low-income and middle-income countries, WHO has convened an effort to develop, evaluate, and illustrate revised risk models. Here, we report the derivation, validation, and illustration of the revised WHO cardiovascular disease risk prediction charts that have been adapted to the circumstances of 21 global regions. METHODS: In this model revision initiative, we derived 10-year risk prediction models for fatal and non-fatal cardiovascular disease (ie, myocardial infarction and stroke) using individual participant data from the Emerging Risk Factors Collaboration. Models included information on age, smoking status, systolic blood pressure, history of diabetes, and total cholesterol. For derivation, we included participants aged 40-80 years without a known baseline history of cardiovascular disease, who were followed up until the first myocardial infarction, fatal coronary heart disease, or stroke event. We recalibrated models using age-specific and sex-specific incidences and risk factor values available from 21 global regions. For external validation, we analysed individual participant data from studies distinct from those used in model derivation. We illustrated models by analysing data on a further 123 743 individuals from surveys in 79 countries collected with the WHO STEPwise Approach to Surveillance. FINDINGS: Our risk model derivation involved 376 177 individuals from 85 cohorts, and 19 333 incident cardiovascular events recorded during 10 years of follow-up. The derived risk prediction models discriminated well in external validation cohorts (19 cohorts, 1 096 061 individuals, 25 950 cardiovascular disease events), with Harrell's C indices ranging from 0·685 (95% CI 0·629-0·741) to 0·833 (0·783-0·882). For a given risk factor profile, we found substantial variation across global regions in the estimated 10-year predicted risk. For example, estimated cardiovascular disease risk for a 60-year-old male smoker without diabetes and with systolic blood pressure of 140 mm Hg and total cholesterol of 5 mmol/L ranged from 11% in Andean Latin America to 30% in central Asia. When applied to data from 79 countries (mostly low-income and middle-income countries), the proportion of individuals aged 40-64 years estimated to be at greater than 20% risk ranged from less than 1% in Uganda to more than 16% in Egypt. INTERPRETATION: We have derived, calibrated, and validated new WHO risk prediction models to estimate cardiovascular disease risk in 21 Global Burden of Disease regions. The widespread use of these models could enhance the accuracy, practicability, and sustainability of efforts to reduce the burden of cardiovascular disease worldwide. FUNDING: World Health Organization, British Heart Foundation (BHF), BHF Cambridge Centre for Research Excellence, UK Medical Research Council, and National Institute for Health Research