Conjugation Approach Research Articles

Hepta-Mutant Staphylococcus aureus Sortase A (SrtA7m) as a Tool for in Vivo Protein Labeling in Caenorhabditis elegans.

In vivo protein ligation is of emerging interest as a means of endowing proteins with new properties in a controlled fashion. Tools to site-specifically and covalently modify proteins with small molecules, peptides, or other proteins in living cells are few and far between. Here, we describe the development of a Staphylococcus aureus sortase (SrtA)-based protein ligation approach for site-specific conjugation of fluorescent dyes and ubiquitin (Ub) to modify proteins in Caenorhabditis elegans. Hepta-mutant SrtA (SrtA7m) expressed in C. elegans is functional and supports in vitro sortase reactions in a low-Ca2+ environment. Feeding SrtA7m-expressing C. elegans with small peptide-based probes such as (Gly)3- biotin or (Gly)3-fluorophores enables in vivo target protein modification. SrtA7m also catalyzes the circularization of suitably modified linear target proteins in vivo and allows the installation of F-box domains on targets to induce their degradation in a ubiquitin-dependent manner. This is a noninvasive method to achieve in vivo protein labeling, protein circularization, and targeted degradation in C. elegans. This technique should improve our ability to monitor and alter the function of intracellular proteins in vivo.

Legomedicine-A Versatile Chemo-Enzymatic Approach for the Preparation of Targeted Dual-Labeled Llama Antibody-Nanoparticle Conjugates.

Conjugation of llama single domain antibody fragments (Variable Heavy chain domains of Heavy chain antibodies, VHHs) to diagnostic or therapeutic nanoparticles, peptides, proteins, or drugs offers many opportunities for optimized targeted cancer treatment. Currently, mostly nonspecific conjugation strategies or genetic fusions are used that may compromise VHH functionality. In this paper we present a versatile modular approach for bioorthogonal VHH modification and conjugation. First, sortase A mediated transPEGylation is used for introduction of a chemical click moiety. The resulting clickable VHHs are then used for conjugation to other groups employing the Cu+-independent strain-promoted alkyne–azide cycloadition (SPAAC) reaction. Using this approach, tail-to-tail bispecific VHHs and VHH-targeted nanoparticles are generated without affecting VHH functionality. Furthermore, this approach allows the bioconjugation of multiple moieties to VHHs for simple and convenient production of VHH-based theranostics.

Enhanced biofilm penetration for microbial control by polyvalent phages conjugated with magnetic colloidal nanoparticle clusters (CNCs)

Biofilm treatment using a polyvalent bacteriophage conjugated with colloidal nanoparticle clusters.

Transgultaminase-Mediated Nanoarmoring of Enzymes by PEGylation.

PEGylation, the covalent attachment of polyethylene glycol to bioactive molecules, is one of the leading approaches used to prolong pharmacokinetics, to improve the stability, and to reduce the immunogenicity of therapeutic proteins. PEG-conjugated products are associated with better therapy outcomes and improved patient compliance. Widely applied in clinical practice, the technology is mainly used to modify proteins, peptides, and oligonucleotides but also other drug delivery systems such as the liposomal one. Undergoing continuous attempts to optimize therapeutic efficacy and to tune the formation of conjugates, a number of different PEGylation processes are now available to researchers for protein conjugation. Although the possibility of obtaining highly homogeneous conjugate mixtures, preferably formed by a single monoconjugate, from a chemical conjugation reaction continues to be limited, several enzymatic conjugation approaches have recently been investigated to address this need. PEGylation mediated by microbial transglutaminase and its many advantages and modifications are outlined in detail in the current work permitting interested readers to perform site-specific protein derivatization to glutamines or lysines.

Polymeric Self-Assemblies with Boron-Containing Near-Infrared Dye Dimers for Photoacoustic Imaging Probes.

Polymers containing pyrrolopyrrole aza-BODIPY (PPAB) and thiophene-bridged BODIPY dimers (TBD) having poly(ethylene glycol) (PEG) or PEGylated hyaluronic acid (HA) were prepared by facile conjugation approaches. Self-assemblies consisting of TBD-conjugated polymers more efficiently generated photoacoustic (PA) signals than PPAB-PEG conjugate upon irradiation with near-infrared pulsed laser light. Among dye-conjugated polymers examined, TBD-HA-PEG conjugates efficiently generated photoacoustic signals, 1.49-1.83 times stronger than that of commercially available indocyanine green (ICG). We found that the following two factors are essential to enhance PA signals from self-assemblies: (1) the formation of strongly interacting TBD aggregates and (2) enhancement of the elastic modulus of self-assemblies by conjugating TBDs with HA. TBD-conjugated HA derivatives circulated in blood vessels for a longer time (15.6 ± 4.9% injected dose (ID) in blood 24 h after injection) and more specifically accumulated in tumor tissues (17.8 ± 3.5% ID/g in tumor 24 h after injection) than ICG-conjugated HA derivatives, visualizing a tumor site more clearly. The cell uptake experiment of dye-HA conjugates indicates that ICG-conjugated polymers internalized into cells or merged with cell walls to emit strong fluorescence, while TBD-conjugated polymers were not internalized into cells. Because the disassembly of the TBD-conjugated HA derivatives is suppressed, aggregated TBDs emit weak fluorescence but efficiently generate strong PA signals in tumor tissues.

Conjugate heat and mass transfer modeling of a new rubber smoking room and experimental validation

A new rubber smoking room has been designed, constructed and tested for natural rubber sheet smoke drying for better air temperature and flow distributions. Three-dimensional numerical model of rubber sheet drying using a conjugate approach is investigated by a computational fluid dynamics simulation. The optimal 2.5m×6.0m×3.5m rubber smoking room contains 40 63.5mm diameter hot gas supply ducts on the floor and four 0.2m×0.2m and one 0.5m×0.5m ventilating lids on the roof. Velocity and temperature of the drying air, and temperature and moisture content of rubber sheets have been predicted. The experimental and simulation results are in good agreement in terms of statistical parameters. R2 for temperature and moisture content are 0.980–0.987 and 0.972, respectively, while RMSE are 5.73–6.99 and 2.20. The drying time is significantly reduced from 72 h in a conventional natural-flow smoking room to 48 h in this new design room.

Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array

Scalable, high-throughput DNA sequencing is a prerequisite for precision medicine and biomedical research. Recently, we presented a nanopore-based sequencing-by-synthesis (Nanopore-SBS) approach, which used a set of nucleotides with polymer tags that allow discrimination of the nucleotides in a biological nanopore. Here, we designed and covalently coupled a DNA polymerase to an α-hemolysin (αHL) heptamer using the SpyCatcher/SpyTag conjugation approach. These porin-polymerase conjugates were inserted into lipid bilayers on a complementary metal oxide semiconductor (CMOS)-based electrode array for high-throughput electrical recording of DNA synthesis. The designed nanopore construct successfully detected the capture of tagged nucleotides complementary to a DNA base on a provided template. We measured over 200 tagged-nucleotide signals for each of the four bases and developed a classification method to uniquely distinguish them from each other and background signals. The probability of falsely identifying a background event as a true capture event was less than 1.2%. In the presence of all four tagged nucleotides, we observed sequential additions in real time during polymerase-catalyzed DNA synthesis. Single-polymerase coupling to a nanopore, in combination with the Nanopore-SBS approach, can provide the foundation for a low-cost, single-molecule, electronic DNA-sequencing platform.

Heat Flow and Solidification Modeling of Industrial Scale, Ingot Casting Operation

A model, based on the concept of effective thermal conductivity, was developed to study thermal fields and the resultant solidification behavior of large, round, industrial size ingots. In this, flow and turbulence phenomena during mold filling as well as subsequent solidification were not modeled explicitly but their influence was accounted for by artificially raising the thermal conductivity of solidifying steel. Thus, a conduction like equation embodying a conjugate approach was applied to simultaneously predict the evolution of temperature fields in the mold as well as in the solidifying ingot following teeming. Prior to comparing model predictions against industrial scale measurements, sensitivity of calculations to grid size, time step height, convergence criterion etc. were rigorously assessed. Similarly, modeling of interfacial resistance, chemical reactions and heat effects in the hot top as well as their influence on predicted results were evaluated computationally. Embodying mixed thermal boundary conditions (free convection + radiation) at the mold wall, temperature fields during solidification of two different industrial large ingots were predicted numerically. Parallely, mold wall temperature was monitored as a function of time and surface temperature of ingot was measured at the instant of mold stripping using hand held, radiation pyrometers. Incorporating relevant operating conditions (viz., mold dimensions and size, ingot and hot top dimensions and material, initial mold and liquid temperature etc.) into the calculation scheme, predictions were made via a computational procedure developed in-house and results thus obtained were compared against equivalent industrial scale measurements. Very reasonable agreement between the two was demonstrated.

Stability Enhancing N-Terminal PEGylation of Oxytocin Exploiting Different Polymer Architectures and Conjugation Approaches.

Oxytocin, a cyclic nine amino acid neurohypophyseal hormone therapeutic, is effectively used in the control of postpartum hemorrhaging (PPH) and is on the WHO List of Essential Medicines. However, oxytocin has limited shelf life stability in aqueous solutions, particularly at temperatures in excess of 25 °C and injectable aqueous oxytocin formulations require refrigeration (<8 °C). This is particularly problematic in the hot climates often found in many developing countries where daytime temperatures can exceed 40 °C and where reliable cold-chain storage is not always achievable. The purpose of this study was to develop N-terminal amine targeted PEGylation strategies utilizing both linear PEG and polyPEG "comb" polymers as an effective method for stabilizing solution formulations of this peptide for prolonged storage in the absence of efficient cold-chain storage. The conjugation chemistries investigated herein include irreversible amine targeted conjugation methods utilizing NHS ester and aldehyde reductive amination chemistry. Additionally, one reversible conjugation method using a Schiff base approach was explored to allow for the release of the native peptide, thus, ensuring that biological activity remains unaffected. The reversibility of this approach was investigated for the different polymer architectures, alongside a nonpolymer oxytocin analogue to monitor how pH can tune native peptide release. Elevated temperature degradation studies of the polymer conjugates were evaluated to assess the stability of the PEGylated analogues in comparison to the native peptide in aqueous formulations to mimic storage conditions in developing nations and regions where storage under appropriate conditions is challenging.

Abstract 2974: Targeting cadherin-6 (CDH6) with an antibody-drug conjugate for the treatment of ovarian and renal cancer

Abstract In an attempt to mine tumor versus normal mRNA expression datasets for novel tumor antigens, we identified the Cadherin-6 (CDH6) gene as frequently overexpressed in ovarian and renal cancers, while featuring a lineage-restricted normal tissue expression pattern. CDH6, also known as K-(kidney)-cadherin, is a member of the cadherin superfamily of calcium-dependent cell-cell adhesion molecules. We hypothesized that based on the combined observation of frequent overexpression of CDH6 in cancer and a restricted normal tissue expression, CDH6 might be an ideal tumor antigen for targeting using an antibody-drug conjugate (ADC) approach. CDH6-ADC is a fully-human anti-CDH6 IgG1, linked via sulfo-SPDB to the maytansinoid payload DM4. The antibody component of CDH6-ADC was selected from a panel of anti-CDH6 antibodies based on a multi-factorial lead selection campaign incorporating readouts of internalization propensity, in vitro cytotoxicity, as well as in vivo PK and efficacy across multiple linker-payload formats. CDH6-ADC features potent, target-dependent in vivo activity consistent with the mechanism of the anti-mitotic, tubulin-targeting sulfo-SPDB-DM4 linker-payload combination used. Specifically, treatment of CDH6-expressing ovarian cancer xenograft models with CDH6-ADC results in the time-dependent generation of intra-tumoral ADC catabolites and concomitant induction of phospho-histone H3 and cleaved caspase-3 - markers of G2/M arrest and apoptosis, respectively. CDH6-ADC induces durable tumor regressions at clinically relevant exposures in multiple human patient-derived tumor xenografts (PDX) across both ovarian and renal cancer lineages. To gain a more thorough understanding of CDH6-ADC activity in pre-clinical models of human ovarian cancer and identify potential molecular correlates for patient stratification, we profiled CDH6-ADC in a PDX clinical trial or PCT comprising 31 individual PDX models. In this unselected population, treatment with CDH6-ADC resulted in robust anti-tumor activity. Integration of PDX response data with CDH6 target expression in both the PDX models and human clinical samples indicate CDH6 expression patterns consistent with in vivo activity are found in a substantial fraction of ovarian, renal and cholangiocarcinoma patients. Together, the encouraging pre-clinical efficacy and tolerability data support the clinical evaluation of CDH6-ADC. Citation Format: Scott D. Collins, Parmita Saxena, Xiao Y. Li, Yeonju Shim, Lance Ostrom, Nicholas C. Yoder, Kalli C. Catcott, Molly A. McShea, Xiuxia Sun, Sanela Bilic, William R. Tschantz, Meghan Flaherty, Keith Mansfield, Tiancen Hu, Vladimir Capka, Markus Kurz, Ivana Liric Rajlic, Anne Serdakowski London, Duc Nguyen, Rebecca Mosher, Matthew J. Meyer, Aaron Bourret, Jamal Saeh, Scott Cameron, Emma Lees, Carl U. Bialucha. Targeting cadherin-6 (CDH6) with an antibody-drug conjugate for the treatment of ovarian and renal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2974.

Abstract 872: In vivo activity of a novel CDH6 targeting antibody-drug conjugate, including population-scale ovarian PDX clinical trial

Abstract The Cadherin-6 (CDH6) gene was found to be frequently overexpressed in ovarian and renal cancers, while featuring a lineage-restricted normal tissue expression pattern. We hypothesized that based on the combined observation of frequent overexpression of CDH6 in cancer and a restricted normal tissue expression, CDH6 might be an ideal tumor antigen for targeting using an antibody-drug conjugate (ADC) approach. CHD6-ADC is a fully-human anti-CDH6 IgG1, linked via sulfo-SPDB to the tubulin-binding maytansinoid payload DM4. CDH6-ADC was evaluated across multiple linker-payload combinations with the sulfo-SPDB-DM4 format being selected based on a superior combined profile pertaining to activity, selectivity and tolerability. To gain a broader understanding of CDH6-ADC activity in vivo we profiled the lead candidate against a panel of 31 unselected patient derived ovarian xenograft (PDX) models in a 1×1×1 PDX clinical trial, similar to that described in Gao et al., 2015. In this unbiased high throughput in vivo screen, CDH6-ADC demonstrated robust antitumor activity, with an overall response rate of 39%. Responses were generally durable beyond 150 days and were achieved at doses yielding exposures anticipated to be achievable in humans and observed in PDX models featuring a range of CDH6 expression level and degree of tumor heterogeneity. Retrospective analysis of individual PDX responses and molecular profiling data demonstrate that sensitivity to CDH6-ADC is highly correlated to CDH6 transcript and protein levels. These findings suggest an ability to prospectively identify patients most likely to benefit from this novel targeted therapy. Furthermore, CDH6-ADC demonstrated robust tumor regressions in a representative PDX xenograft model that was refractory to carboplatin/paclitaxel standard of care therapy. These data suggest that CDH6-ADC may benefit both treatment naïve patients and patients that have progressed on prior therapy containing tubulin-targeting anti-mitotics. Extending beyond ovarian cancer, we found CDH6 to be frequently overexpressed in renal cancer. CDH6-ADC was active against RCC PDX models featuring patient relevant levels of CDH6 expression. Data described herein suggest that this novel ADC may be an effective treatment for patients with CDH6 expressing tumors, including ovarian and renal cancer - both indications with a high unmet medical need. Citation Format: Carl U. Bialucha, Scott D. Collins, Yeonju Shim, Xiamei Zhang, Roberto Velazquez, Colleen Kowal, Caroline Bullock, Hongbo Cai, Stacy M. Rivera, Julie M. Goldovitz, Esther Kurth, Alice T. Loo, Guizhi Yang, John Green, Lance Ostrom, Matthew J. Meyer, Rebecca Mosher, Hui Gao, Juliet Williams, Emma Lees. In vivo activity of a novel CDH6 targeting antibody-drug conjugate, including population-scale ovarian PDX clinical trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 872.

Conjugation of Docetaxel with Multiwalled Carbon Nanotubes and Codelivery with Piperine: Implications on Pharmacokinetic Profile and Anticancer Activity.

Nanotechnology-based drug products are emerging as promising agents to enhance the safety and efficacy of established chemotherapeutic molecules. Carbon nanotubes (CNTs), especially multiwalled CNTs (MWCNTs), have been explored for this potential owing to their safety and other desired attributes. Docetaxel (DTX) is an indispensable anticancer agent, which has wide applicability in variety of cancers. However, the potential of DTX is still not completely harvested due to problems like poor aqueous solubility, low tissue permeability, poor bioavailability, high first pass metabolism, and dose-related toxicity. Hence, it was proposed to attach DTX to MWCNTs and coadminister it along with piperine with an aim to enhance the tissue permeation, anticancer activity, and bioavailability. The Fourier transform infrared, UV, and NMR spectroscopic data confirmed successful conjugation of DTX to MWCNTs and adsorption of piperine onto MWCNTs. The codelivery MWCNT-based system offered drug release moderation and better cancer cell toxicity than that of plain DTX as well as DTX-CNT conjugate. The pharmacokinetic profile of DTX was exceptionally improved by the conjugation, in general, and coadministration with piperine, in specific vis-à-vis plain drug. Hence, the dual approach of MWCNTs conjugation and piperine coadministration can serve as a beneficial option for enhancement of the performance of DTX in cancer chemotherapy.

Shape-Encoded Chitosan-Polyacrylamide Hybrid Hydrogel Microparticles with Controlled Macroporous Structures via Replica Molding for Programmable Biomacromolecular Conjugation.

Polymeric hydrogel microparticle-based suspension arrays with shape-based encoding offer powerful alternatives to planar and bead-based arrays toward high throughput biosensing and medical diagnostics. We report a simple and robust micromolding technique for polyacrylamide- (PAAm-) based biopolymeric-synthetic hybrid microparticles with controlled 2D shapes containing a potent aminopolysaccharide chitosan as an efficient conjugation handle uniformly incorporated in PAAm matrix. A postfabrication conjugation approach utilizing amine-reactive chemistries on the chitosan shows stable incorporation and retained chemical reactivity of chitosan, readily tunable macroporous structures via simple addition of low content long-chain PEG porogens for improved conjugation capacity and kinetics, and one-pot biomacromolecular assembly via bioorthogonal click reactions with minimal nonspecific binding. We believe that the integrated fabrication-conjugation approach reported here could offer promising routes to programmable manufacture of hydrogel microparticle-based biomacromolecular conjugation and biofunctionalization platforms for a large range of applications.

Prospects of Bacteriotherapy with Nanotechnology in Nanoparticledrug Conjugation Approach for Cancer Therapy.

Bacteriotherapy and nanotechnology have shown remarkable potential in diagnostic and therapeutic applications for various diseases. Individual impacts of these micro-nano systems over different aspects of human health are well studied; however, an integrated system of bacteria-nanoparticle (NP) conjugation is less explored. The untamed potential of bacteria-NP conjugation could be a new tool for diagnosis and treatment of invasive diseases like malaria, tuberculosis and cancer. Mammalian cells exhibit cytosis as their defense mechanism when they encounter foreign elements such as bacteria. In these mammalian cells, during phagocytosis, bacteria are ruptured and lysed by lysozymes. A bacterium carrying the drug-tagged NP would be engulfed in the same manner and ultimately reaches the target cells. Rapid and continuous cell divisions in the cancer tissues lead to defective vessels, underdeveloped cellcell interconnects, development of hypoxic areas and heterogeneous population of tumor cells. This unorganized and poorly developed angiogenesis in tumor cells makes it difficult for conventional chemotherapeutic drugs to localize the tumors selectively. In the present scenario of diagnosis and treatment of cancer/tumor cells, it could be expected that the existing bacteriotherapy with the advanced nanotechnology would be a way further in the targeted drug delivery for cancer therapy. This review emphasizes the potential applications of bacteriotherapy with nanotechnology for the diagnosis and treatment of cancer.

Functionalization of Polydopamine via the Aza-Michael Reaction for Antimicrobial Interfaces.

Polydopamine (pDA) coatings afford tremendous versatility due to their capabilities to provide substrate-independent functionalization with a wide range of amine- and thiol-containing molecules. In this work, we developed a new and facile conjugation approach to the formation of β-amino carbonyl linkages between pDA and acrylate/acrylamide molecules via the aza-Michael reaction. Sulfobetaine acrylamide (SBAA), sulfobetaine methacrylate (SBMA), and poly(ethylene glycol) methacrylate (PEGMA) were employed to graft onto pDA films, giving rise to formation of antifouling coatings. Because of the universal adhesive property of pDA, the coating strategy was applied to different substrates, including TiO2, gold, SiO2, Nitinol alloy, polystyrene, and poly(dimethylsiloxane). The variation of surface chemistry and surface wettability upon pDA modification and subsequent conjugation was monitored with X-ray photoelectron spectroscopy (XPS) and water contact angle measurements. Antifouling properties of coatings were challenged by three common Gram-negative and Gram-positive bacteria. Cytocompatibility of the coatings with NIH-3T3 fibroblasts was accessed using MTT assay. The results showed that pDA coatings grafted with SBAA exhibited superhydrophilicity and excellent fouling resistance likely due to the high chemical reactivity of acrylamide, leading to high grafting density. In addition, dual functional coatings containing passive and active antibacterial components were constructed through the in situ deposition of antimicrobial agent, silver nanoparticles, in pDA, followed by the grafting of SBAA for bacterial repellence. The composite coatings allowed reducing adsorption of E.coli by >95%, while killing attached bacteria by up to 98% upon the releasing of Ag(+) ions as measured by inductively coupled plasma mass spectrometry. Consequently, this work paves a new avenue to the grafting strategy to engineer pDA and to the functional bioinspired antifouling interfaces in a substrate-independent fashion.

Gd(III)-Dithiolane Gold Nanoparticles for T1-Weighted Magnetic Resonance Imaging of the Pancreas.

Pancreatic adenocarcinoma has a 5 year survival of approximately 3% and median survival of 6 months and is among the most dismal of prognoses in all of medicine. This poor prognosis is largely due to delayed diagnosis where patients remain asymptomatic until advanced disease is present. Therefore, techniques to allow early detection of pancreatic adenocarcinoma are desperately needed. Imaging of pancreatic tissue is notoriously difficult, and the development of new imaging techniques would impact our understanding of organ physiology and pathology with applications in disease diagnosis, staging, and longitudinal response to therapy in vivo. Magnetic resonance imaging (MRI) provides numerous advantages for these types of investigations; however, it is unable to delineate the pancreas due to low inherent contrast within this tissue type. To overcome this limitation, we have prepared a new Gd(III) contrast agent that accumulates in the pancreas and provides significant contrast enhancement by MR imaging. We describe the synthesis and characterization of a new dithiolane-Gd(III) complex and a straightforward and scalable approach for conjugation to a gold nanoparticle. We present data that show the nanoconjugates exhibit very high per particle values of r1 relaxivity at both low and high magnetic field strengths due to the high Gd(III) payload. We provide evidence of pancreatic tissue labeling that includes MR images, post-mortem biodistribution analysis, and pancreatic tissue evaluation of particle localization. Significant contrast enhancement was observed allowing clear identification of the pancreas with contrast-to-noise ratios exceeding 35:1.

A taxonomy of mixing and outcome distributions based on conjugacy and bridging

The generalized linear mixed model (GLMM) is commonly used for the analysis of hierarchical non Gaussian data. It combines an exponential family model formulation with normally distributed random effects. A drawback is the difficulty of deriving convenient marginal mean functions with straightforward parametric interpretations. Several solutions have been proposed, including the marginalized multilevel model (directly formulating the marginal mean, together with a hierarchical association structure) and the bridging approach (choosing the random-effects distribution such that marginal and hierarchical mean functions share functional forms). Another approach, useful in both a Bayesian and a maximum-likelihood setting, is to choose a random-effects distribution that is conjugate to the outcome distribution. In this paper, we contrast the bridging and conjugate approaches. For binary outcomes, using characteristic functions and cumulant generating functions, it is shown that the bridge distribution is unique. Self-bridging is introduced as the situation in which the outcome and random-effects distributions are the same. It is shown that only the Gaussian and degenerate distributions have well-defined cumulant generating functions for which self-bridging holds.

Phase Transfer and Surface Functionalization of Hydrophobic Nanoparticle using Amphiphilic Poly(amino acid).

Functionalization of nanoparticles with chemical and biochemical is essential for their biomedical and other application. However, most of the high quality nanoparticles are hydrophobic in nature due to surfactant capping and their conversion into water-soluble functional nanoparticle via appropriate coating and conjugation chemistry is extremely critical issue. Here we report amphiphilic poly(amino acid)-based one-pot coating and conjugation approach that can transform hydrophobic nanoparticle into water-soluble nanoparticle functionalized with primary amine, thiol, and biomolecule. We have designed amphiphilic polyaspartimide that can anchor hydrophobic nanoparticle through octadecyl groups, leaving the polar polyethylene glycol and aspartimide groups exposed outwards. The aspartimide group is then reacted with primary amine containing chemical/biomolecule with the formation of water-soluble functional nanoparticle. This approach has been extended to different hydrophobic nanoparticles and biomolecules. The present approach has advantages over existing approaches as coating and functionalization can be performed in one pot and functional nanoparticles have <12 nm hydrodynamic size, high colloidal stability, and biocompartibility. This developed approach can be used to derive biocompatible nanobioconjugates for various biomedical applications.

Dendrimer-drug Conjugates in Drug Delivery and Targeting

Background: Conjugation of several bio-actives to macromolecular nanocarriers is a promising approach to improve the efficiency and reduce side effects. Dendrimer represents a novel class of synthetic nanocarriers, comprised of different branched materials with tailor-made functionalities to conjugate wide varieties of bioactive molecules, therapeutic agents and different pharmacological substances including antibodies, ligands etc. Such opportunistic properties has been equally explored for the purpose of diagnostic applications as well. Methods: We have accomplished thorough bibliographic search and compilation of referred/indexed peer-reviewed literature to concisely review the above mentioned concept. The organized study was carried out concerned for dendrimer-drug conjugates employed in drug delivery and targeting to specific sites for their biological and clinical evaluation. Results: One hundred forty one papers were referred in this review after throughout literature survey. The conjugation is favorable and is easy to perform with the carriers like dendrimers due to its unique properties and multiple functional groups at the surface. The present review highlights the general properties class and applications of dendrimers in drug delivery. The focus is particularly on the dendrimer drug conjugates which are comparatively less explored area than the non-covalent dendrimer drug interactions. Conclusion: Conjugation of drugs to the dendrimers has particularly resulted into the increased drug payload and improved drug activity as evidenced by the reports discussed here in this article. Therefore in future it would be more advantageous to explore the dendrimer drug conjugation approaches for delivering drugs through dendrimers. Keywords: Conjugation, dendrimer, drug delivery, nanocarrier, targeting.

Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II).

BackgroundThe ability to site-specifically conjugate a protein to a payload of interest (e.g., a fluorophore, small molecule pharmacophore, oligonucleotide, or other protein) has found widespread application in basic research and drug development. For example, antibody-drug conjugates represent a class of biotherapeutics that couple the targeting specificity of an antibody with the chemotherapeutic potency of a small molecule drug. While first generation antibody-drug conjugates (ADCs) used random conjugation approaches, next-generation ADCs are employing site-specific conjugation. A facile way to generate site-specific protein conjugates is via the aldehyde tag technology, where a five amino acid consensus sequence (CXPXR) is genetically encoded into the protein of interest at the desired location. During protein expression, the Cys residue within this consensus sequence can be recognized by ectopically-expressed formylglycine generating enzyme (FGE), which converts the Cys to a formylglycine (fGly) residue. The latter bears an aldehyde functional group that serves as a chemical handle for subsequent conjugation.ResultsThe yield of Cys conversion to fGly during protein production can be variable and is highly dependent on culture conditions. We set out to achieve consistently high yields by modulating culture conditions to maximize FGE activity within the cell. We recently showed that FGE is a copper-dependent oxidase that binds copper in a stoichiometric fashion and uses it to activate oxygen, driving enzymatic turnover. Building upon that work, here we show that by supplementing cell culture media with copper we can routinely reach high yields of highly converted protein. We demonstrate that cells incorporate copper from the media into FGE, which results in increased specific activity of the enzyme. The amount of copper required is compatible with large scale cell culture, as demonstrated in fed-batch cell cultures with antibody titers of 5 g · L−1, specific cellular production rates of 75 pg · cell−1 · d−1, and fGly conversion yields of 95–98 %.ConclusionsWe describe a process with a high yield of site-specific formylglycine (fGly) generation during monoclonal antibody production in CHO cells. The conversion of Cys to fGly depends upon the activity of FGE, which can be ensured by supplementing the culture media with 50 uM copper(II) sulfate.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0254-0) contains supplementary material, which is available to authorized users.