Automated PET-RAFT Polymerization Towards Pharmaceutical Amorphous Solid Dispersion Development.

In pharmaceutical oral drug delivery development, about 90% of drugs in the pipeline have poor aqueous solubility leading to severe challenges with oral bioavailability and translation to effective and safe drug products. Amorphous solid dispersions (ASDs) have been utilized to enhance the oral bioavailability of poorly soluble active pharmaceutical ingredients (APIs). However, a limited selection of regulatory-approved polymer excipients exists for the development and further understanding of tailor-made ASDs. Thus, a significant need exists to better understand how polymers can be designed to interact with specific API moieties. Here, we demonstrate how an automated combinatorial library approach can be applied to the synthesis and screening of polymer excipients for the model drug probucol. We synthesized a library of 25 random heteropolymers containing one hydrophilic monomer (2-hydroxypropyl acrylate (HPA)) and four hydrophobic monomers at varied incorporation. The performance of ASDs made by a rapid film casting method was evaluated by dissolution using ultra-performance liquid chromatography (UPLC) sampling at various time points. This combinatorial library and rapid screening strategy enabled us to identify a relationship between polymer hydrophobicity, monomer hydrophobic side group geometry, and API dissolution performance. Remarkably, the most effective synthesized polymers displayed slower drug release kinetics compared to industry standard polymer excipients, showing the ability to modulate the drug release profile. Future coupling of high throughput polymer synthesis, high throughput screening (HTS), and quantitative modeling would enable specification of designer polymer excipients for specific API functionalities.

Purifying Low-Volume Combinatorial Polymer Libraries with Gel Filtration Columns.

Recent advances in oxygen-tolerant controlled/living radical polymer chemistry now enable efficient synthesis of diverse and combinatorial polymer libraries. While library synthesis has been dramatically simplified, equally efficient purification strategies for removal of small-molecule impurities are not yet established in high throughput settings. It is shown that gel filtration columns for chromatography frequently used in the protein science community are well suited for high throughput polymer purification. Using either single-use columns or gel filtration plates, the purification of 32 diverse polymers is demonstrated in a library with >95% removal of small molecule impurities and >85% polymer retention in a single purification step. Doing so replaces the typical procedure of polymer precipitation, which requires solvent optimization for each polymer in a complex library. Overall, this work raises awareness in the polymer science community that gel filtration is amenable to purification of large polymer libraries and can speed up the progress of combinatorial polymer chemistry.

Synergistic effect of silver nitrate and coconut water on shoot differentiation and plant regeneration from cultured cotyledons of Capsicum annuum L.

Chili pepper (Capsicum annuum L.) ranks among the most important vegetable crop belonging to the family Solanaceae that is consumed both as vegetable and spice throughout the world. C. annuum, as crop, in order to meet the target yield, demands improved variety that could overcome environmental challenges viz., biotic and abiotic stress. Cultivar improvement essentially requires an efficient in vitro regeneration protocol. In the present study, we investigated the influence of silver nitrate (AgNO3) and coconut water, individually as well in combination, on in vitro shoot elongation and plant regeneration from cotyledon explants of C. annuum cv G-4. Shoot buds were induced on shoot bud induction medium supplemented with either 44.38 µM 6-benzylaminopurine (BAP) or 9.0 µM thidiazuron (TDZ) along with 5.77 µM gibberellic acid (GA3) and 14.7 µM phenyl acetic acid (PAA). Elongation of shoot buds was obtained on elongation medium containing 8.87 µM BA or 0.45 µM TDZ, 5.77 µM GA3 and 14.7 µM PAA followed by rooting in 9.8 µM indole-3-butyric acid (IBA). All the media were supplemented with 30 µM AgNO3 and/or coconut water (10% v/v). The presence of coconut water in the elongation media enhanced the regeneration of well developed shoots from differentiating explants on TDZ media while AgNO3 resulted in enhanced production of rooted shoots with greater influence on emerging shoots from BAP media upon transfer to rooting media. There was synergistic response with further enhancement of elongated shoots/elongated rooted shoots on the combined use of coconut water and AgNO3. The elongation media produced significantly higher total shoots when AgNO3 was used synergistically with coconut water (59.0%) as against AgNO3 alone (38.0%). While in rooting media, there was significantly higher production of elongated rooted shoots when coconut water was used synergistically with AgNO3 (47.2%) as against the coconut water alone (14.4%).

Differential response of tomato and tobacco to Agrobacterium mediated transformation with cytokinin independent -1 (CKI-1) gene as influenced by cytokinin levels.

Cytokinin independent-1 (CKI-1) gene was identified through its ability to confer cytokinin independent growth in Arabidopsis which has led to this gene being advocated as a selectable marker in plant transformation. Keeping this in view, CKI-1 gene was assessed as a selectable marker by transformation of tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum L.). Overexpression of CKI-1 gene through Agrobacterium mediated transformation in tobacco and tomato conferred cytokinin independent shoot regeneration (in media devoid of cytokinin/plant growth regulators) in tobacco, but not in tomato wherein this ability (cytokinin independence) was conferred to T1 explants of CKI-1 transgenic tomato plant (T0) regenerated on cytokinin medium. Analysis of cytokinin levels revealed that cytokinin independent growth upon transformation with CKI-1 gene in tobacco (T0) and tomato (T1) was achieved through maintaining/regulating higher endogenous cytokinin levels and CKI-1 gene expression. Levels of CKI-1 transcripts assayed through quantitative RT-PCR suggested that there seemed to be a threshold level of endogenous cytokinin level, regulated due to external or internal supply via CKI-1 gene upto which CKI-1 gene expression correlated with endogenous cytokinin content and beyond that, either the gene expression was not induced or it remains same. With the incorporation of CKI-1 gene, it appeared that this threshold level of endogenous cytokinin might be reduced in crops like tomato to support shoot regeneration at lower concentration of cytokinin, but could not be made independent of external supply of cytokinin as in tobacco suggesting that use of CKI-1 gene as an effective alternate selection marker could not be universally applicable across the species. The results of the present study revealed that CKI-1 gene in addition to enhancing cytokinin levels, was also involved in contributing to the sensitivity to cytokinin and thus served as a positive regulator of cytokinin signal transduction.

Ultrastructural characterization of the implant/bone interface of immediately loaded dental implants.

Primary stability and an optimized load transfer are assumed to account for an undisturbed osseointegration process of implants. Immediate loaded newly designed titanium dental implants inserted in the mandible of minipigs were used for the characterization of the interfacial area between the implant surface and the surrounding bone tissue during the early healing phase. Histological and electron microscopical studies were performed from implant containing bone specimens. Two different load regimens were applied to investigate the load related tissue reaction. Histological and electron microscopical analysis revealed a direct bone apposition on the implant surfaces, as well as the attachment of cells and matrix proteins in the early loading phase. A striking finding of the ultrastructural immunocytochemical investigations was the synthesis and deposition of bone related proteins (osteonectin, fibronectin, fibronectin receptor) by osteoblasts from day one of bone/biomaterial interaction. Calcium-phosphate needle-like crystallites were newly synthesized in a time-related manner directly at the titanium surface. No difference in the ultrastructural appearance of the interface was found between the two loading groups. Our experimental data suggest that loading of specially designed implants can be performed immediately after insertion without disturbing the biological osseointegration process.

A novel method of isolation of a bone-morphogenetic-protein-like protein from ossein.

A new bone-morphogenetic-protein (BMP)-like protein has been isolated through a new protocol from a novel source, ossein. The BMP-like protein was hydrophilic and characterized through Fourier-transform IR studies, SDS/PAGE and coupled with a neutral binder, hydroxypropylmethylcellulose (HPMC) for control release. The IR spectrum of the protein showed peaks in tandem with BMP from bone matrix, and its molecular mass was in the range 18-21 kDa. Sustained release from the surface of HPMC was achieved for a period of 3 days.

Preparation and characterization of a new bioinorganic composite: collagen and hydroxyapatite.

Studies on biomineralization in bone have opened new arenas in the field of fabrication and engineering of bone-graft devices. A new bioinorganic composite was prepared by introducing processed collagenous matrices into the calcifying solutions at pH 7.8-8.0. The calcifying solutions were prepared by using a saturated dicalcium salt solution. Hydroxyapatite grew on the matrix over a period of 3-5 days in a specially designed crystallizing chamber. Changes in biochemical characters and biophysical parameters have given interesting insights into factors affecting and effecting initiation and progression of calcification on organic matrices.