Towards machine learning for hydrogel drug delivery systems

Hydrogel drug delivery system development is complex and laborious, and machine learning (ML) techniques hold great promise in accelerating the process. We highlight recent advances and strategies for data collection and ML, and we discuss the potential for and barriers to the broader use of ML for hydrogel drug delivery systems.     

Heatable carbon nanotube composite membranes for sustainable recovery from biofouling

Membrane filtration is one of the most reliable methods for water treatment. However, wider application is limited due to biofouling caused by accumulation of microorganisms on the membrane surface. This report details a heatable carbon nanotube composite membrane with self-cleaning properties for sustainable recovery from biofouling. Microfiltration polycarbonate/carbon-nanotubes hybrid membranes were fabricated using drawable nanotubes that maintained the porosity and provided electrical conductivity to the membrane. Less than 25 V potential and 2–3 W power increase membrane temperature to 100°C in ~10 s. This temperature is above what most microbial life, bacteria and viruses can handle. When this membrane was employed, filtered Escherichia coli collected on its surface were successfully annihilated within 1 min. Ohmic heating of this membrane could be an effective solution to combat biofouling and complications associated with membrane-based filtration. This is a novel and highly desirable approach to combat biofouling, due to its simplicity and economic advantage.     

Caged protein nanoparticles for drug delivery

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Secretins: dynamic channels for protein transport across membranes

Secretins form megadalton bacterial-membrane channels in at least four sophisticated multiprotein systems that are crucial for translocation of proteins and assembled fibers across the outer membrane of many species of bacteria. Secretin subunits contain multiple domains, which interact with numerous other proteins, including pilotins, secretion-system partner proteins, and exoproteins. Our understanding of the structure of secretins is rapidly progressing, and it is now recognized that features common to all secretins include a cylindrical arrangement of 12-15 subunits, a large periplasmic vestibule with a wide opening at one end and a periplasmic gate at the other. Secretins might also play a key role in the biogenesis of their cognate secretion systems.     

A protein chimera self-assembling unit for drug delivery

In the modern view of selective drug delivery of bioactive molecules, the attention is moving onto the setup of the perfect carrier more than in the optimization of the active compound. In this respect, virus-like particles constitute bioinspired nanodevices with the intrinsic ability to transport a large class of molecules, ranging from smart drugs to small interfering RNAs. In this work, we demonstrate the efficacy of a novel construct obtained by fusing a self-assembling protein from the human Rotavirus A, VP6, with the Small Ubiquitin Modifier domain, which maintains the ability to form nanoparticles and nanotubes and is able to be used as a drug carrier, even without specific targeting epitopes. The high expression and purification yield, combined with low toxicity of the empty particles, clearly indicate a good candidate for future studies of selective drug delivery. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2769, 2019.     

Synthesis and physicochemical characterization of folate-cyclodextrin bioconjugate for active drug delivery

beta-Cyclodextrin-poly(ethylene glycol)-folic acid conjugate (CD-PEG-FA) was synthesized according to a two-step procedure: (1). synthesis of CD-PEG-NH(2) by reaction of monotosyl-activated beta-cyclodextrin with excess of 700 Da diamino-PEG; (2). synthesis of CD-PEG-FA by reaction of CD-PEG-NH(2) with succinimidyl ester-activated folic acid. The CD-PEG-NH(2) intermediate was purified by precipitation in acetone, and the CD-PEG-FA by gel permeation and C-18 reversed-phase chromatography. Both CD-PEG-NH(2) and CD-PEG-FA were analyzed by mass spectrometry, (1)H NMR, and UV-vis spectroscopy. All analytical methods confirmed the theoretical composition of the conjugates: the CD-PEG-NH(2) intermediate was composed of CD and PEG in the molar ratio of 1:1, and the CD-PEG-FA was composed of beta-cyclodextrin, PEG, and folic acid in the molar ratio of 1:1:1. The CD-PEG-FA conjugate was highly soluble in buffer (>42 mM) as compared to the unmodified beta-cyclodextrin (16.3 mM). Phase solubility diagrams of beta-estradiol revealed that drug solubility increases from 11 microM in buffer to 600 microM in the presence of beta-cyclodextrins and 5900 microM with CD-PEG-FA. However, the affinity of beta-estradiol for beta-cyclodextrins decreased about 4 times with PEG and folic acid conjugation. Stability studies carried out using chlorambucil confirmed that the conjugate partially prevents drug degradation in buffer, although this effect was considerably lower than that obtained with beta-cyclodextrin. Computer modeling studies showed that the folic acid linked to the beta-cyclodextrins through a PEG spacer could partially interact with the cyclodextrin cavity. Finally, CD-PEG-FA displayed reduced hemolytic effect as compared to unmodified beta-cyclodextrin.     

Performance characteristics of nanoporous carbon membranes for protein ultrafiltration

Nanoporous carbon membranes could be very attractive for applications of ultrafiltration in the biotechnology industry because of their greater mechanical strength and longer membrane life. The objective of this study was to obtain quantitative data on the performance characteristics of nanoporous carbon membranes formed within a stainless steel support that was first modified by deposition of silica particles within the macroporous support. The nanoporous carbon membrane effectively removed small solutes from a protein solution using diafiltration, with performance comparable to that of commercial polymeric membranes. Protein fouling was evident, although the nanoporous carbon membranes were easily regenerated; cleaning with 0.5 N NaOH at 50 degrees C completely restored the water permeability for multiple cycles. The nanoporous carbon membranes were also compatible with steam sterilization. Significant increases in process flux could be obtained using periodic back-pulsing, with no evidence of any structural alterations in the membrane. These results clearly demonstrate the potential benefits and opportunities for using nanoporous carbon membranes for protein ultrafiltration.     

Multifunctional polymeric micelles with folate-mediated cancer cell targeting and pH-triggered drug releasing properties for active intracellular drug delivery

A new type of multifunctional polymeric micelle drug carrier for active intracellular drug delivery was prepared and characterized in this study. The micelle is a nano-supramolecular assembly with a spherical core-shell structure, and its surface and core were modified with piloting molecules for cancer cells and pH-sensitive drug binding linkers for controlled drug release, respectively. In order to prepare such micelles, self-assembling amphiphilic block copolymers, folate-poly(ethylene glycol)-poly(aspartate hydrazone adriamycin) [Fol-PEG-P(Asp-Hyd-ADR)], were specially designed and synthesized by installing a molecular promoter to enhance intracellular transport, folate (Fol), at the end of the shell-forming PEG chain and conjugating the anticancer drug, adriamycin (ADR), to the side chain of the core-forming PAsp segment through an acid-sensitive hydrazone bond. Because folate-binding proteins (FBP) are selectively overexpressed on the cancer cell membranes, the folate-bound micelles (FMA) can be guided to the cancer cells in the body, and after the micelles enter the cells, hydrazone bonds are cleaved by the intracellular acidic environment (pH 5-6) so that the drug release profile of the micelles is controlled pH-dependently. In this regard, FBP-binding selectivity of the prepared FMA was evaluated by surface plasmon resonance (SPR) measurements. The tetrazolium dye method (MTT assay) using human pharyngeal cancer cells (KB cell) revealed that FMA significantly improved cell growth inhibitory activity in spite of a short exposure time due to the selective and strong interaction between folate molecules and their receptors. Subsequent flow cytometric analysis showed that cellular uptake of FMA significantly increased. Consequently, these findings would provide one of the most effective approaches for cancer treatment using intracellular environment-targeting supramolecular drug carriers.     

Adapter protein for site-specific conjugation of payloads for targeted drug delivery

High-affinity interactions of two fragments of human RNase I (1-15-aa Hu-tag and 21-125-aa HuS adapter protein) can be used for assembly of targeting drug delivery complexes. In this approach, a targeting protein is expressed as a fusion protein with a 15-aa Hu-tag, while HuS is conjugated to a drug (or a drug carrier) creating a "payload" module, which is then bound noncovalently to the Hu-tag of the targeting protein. Although this approach eliminates chemical modifications of targeting proteins, the payload modules are still constructed by random cross-linking of drugs or drug carriers to an adapter protein that might lead to functional heterogeneity of the complexes. To avoid this problem, we engineered an adapter protein HuS(N88C) with an unpaired cysteine in position 88 that can be directly modified without interference with activity of assembled targeting complexes. HuS(N88C) binds Hu-tagged annexin V with K(D) of 50 +/- 6 nM, which is comparable to that of wild-type HuS. To demonstrate the utility of HuS(N88C) for developing uniform payload modules, we constructed a HuS(N88C)-lipid conjugate and inserted it into preformed liposomes loaded with a fluorescent dye. Targeting proteins, Hu-tagged vascular endothelial growth factor or Hu-tagged annexin V, were docked to liposomes decorated with HuS, and the assembled complexes delivered liposomes selectively to target cells.     

Polymeric micelles with water-insoluble drug as hydrophobic moiety for drug delivery

The hydrophobic block of polymeric micelles formed by amphiphilic copolymers has no direct therapeutical effect, and the metabolites of these hydrophobic segments might lead to some unexpected side effects. Here the hydrophobic core of polymeric micelles is replaced by highly water-insoluble drugs themselves, forming a new micellar drug delivery system. By grafting hydrophobic drugs of paclitaxel (PTX) onto the surface of hydrophilic hyperbranched poly(ether-ester) (HPEE), we constructed an amphiphilic copolymer (HPEE-PTX). HPEE-PTX could self-assemble into micellar nanoparticles in aqueous solution with tunable drug contents from 4.1 to 10.7%. Moreover, the hydrolysis of HPEE-PTX in serum resulted in the cumulative release of PTX. In vivo evaluation indicated that the dosage toleration of PTX in mice had been improved greatly and HPEE-PTX micellar nanoparticles could be used as an efficient prodrug with satisfactory therapeutical effect. We believe that most of the lipophilic drugs could improve their characters through this strategy.     

Flexible polyimide probes with microelectrodes and embedded microfluidic channels for simultaneous drug delivery and multi-channel monitoring of bioelectric activity

The study of intracellular communication requires devices that can not only monitor the bioelectric activity, but also control and observe the biochemical environment at the cellular level. This paper reports on the development and characterisation of implantable polyimide microprobes that allow simultaneous, selective chemical delivery/probing and multi-channel recording/stimulation of bioelectric activity. The key component of the system is a flexible polyimide substrate with embedded microchannels that is batch-fabricated combining polyimide micromachining and a lamination technique. The devices provide platinum microelectrodes on both sides of the polyimide substrate with an active surface between 20 microm x 20 microm and 50 microm x 50 microm. The embedded microchannels permit highly localised drug delivery or probing at the tip of the device via channel outlets adjacent to the microelectrodes. The microelectrodes were characterised by electrical impedance spectroscopy and the microchannels were studied in microflow experiments. Two different fluid delivery schemes were explored in two different designs. The first device type consists of a simple combination of microchannels and microelectrodes on one substrate. Liquids are ejected at the tip of the device by pressure injection techniques. The second device was inspired by the so-called U-tube concept allowing for highly localised delivery of controlled amounts of liquids in the picoliters range. Thus, the influence of chemical compounds on the electrical activity of cells can be studied with high temporal and spatial resolution. The flexible, implantable devices can be used for studying the chemical and electrical information exchange and communication of cells in in vivo and in vitro experiments.     

Biosafety of non-surface modified carbon nanocapsules as a potential alternative to carbon nanotubes for drug delivery purposes

Background

Carbon nanotubes (CNTs) have found wide success in circuitry, photovoltaics, and other applications. In contrast, several hurdles exist in using CNTs towards applications in drug delivery. Raw, non-modified CNTs are widely known for their toxicity. As such, many have attempted to reduce CNT toxicity for intravenous drug delivery purposes by post-process surface modification. Alternatively, a novel sphere-like carbon nanocapsule (CNC) developed by the arc-discharge method holds similar electric and thermal conductivities, as well as high strength. This study investigated the systemic toxicity and biocompatibility of different non-surface modified carbon nanomaterials in mice, including multi-walled carbon nanotubes (MWCNTs), single-walled carbon nanotubes (SWCNTs), carbon nanocapsules (CNCs), and C₆₀ fullerene (C₆₀). The retention of the nanomaterials and systemic effects after intravenous injections were studied.

Methodology and Principal Findings

MWCNTs, SWCNTs, CNCs, and C₆₀ were injected intravenously into FVB mice and then sacrificed for tissue section examination. Inflammatory cytokine levels were evaluated with ELISA. Mice receiving injection of MWCNTs or SWCNTs at 50 µg/g b.w. died while C₆₀ injected group survived at a 50% rate. Surprisingly, mortality rate of mice injected with CNCs was only at 10%. Tissue sections revealed that most carbon nanomaterials retained in the lung. Furthermore, serum and lung-tissue cytokine levels did not reveal any inflammatory response compared to those in mice receiving normal saline injection.

Conclusion

Carbon nanocapsules are more biocompatible than other carbon nanomaterials and are more suitable for intravenous drug delivery. These results indicate potential biomedical use of non-surface modified carbon allotrope. Additionally, functionalization of the carbon nanocapsules could further enhance dispersion and biocompatibility for intravenous injection.     

Engineering protein self-assembling in protein-based nanomedicines for drug delivery and gene therapy

Lack of targeting and improper biodistribution are major flaws in current drug-based therapies that prevent reaching high local concentrations of the therapeutic agent. Such weaknesses impose the administration of high drug doses, resulting in undesired side effects, limited efficacy and enhanced production costs. Currently, missing nanosized containers, functionalized for specific cell targeting will be then highly convenient for the controlled delivery of both conventional and innovative drugs. In an attempt to fill this gap, health-focused nanotechnologies have put under screening a growing spectrum of materials as potential components of nanocages, whose properties can be tuned during fabrication. However, most of these materials pose severe biocompatibility concerns. We review in this study how proteins, the most versatile functional macromolecules, can be conveniently exploited and adapted by conventional genetic engineering as efficient building blocks of fully compatible nanoparticles for drug delivery and how selected biological activities can be recruited to mimic viral behavior during infection. Although engineering of protein self-assembling is still excluded from fully rational approaches, the exploitation of protein nano-assemblies occurring in nature and the direct manipulation of protein–protein contacts in bioinspired constructs open intriguing possibilities for further development. These methodologies empower the construction of new and potent vehicles that offer promise as true artificial viruses for efficient and safe nanomedical applications.     

Multiscale modeling of protein membrane interactions for nanoparticle targeting in drug delivery

The objective is to develop easily computable and physiologically predictive mechanism-based multiscale pharmacodynamic models for targeted drug delivery.

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The covalent bioconjugate of multiwalled carbon nanotube and amino‐modified linearized plasmid DNA for gene delivery

Carbon nanotubes (CNTs) are allotropes of carbon, which have unique physical, mechanical, and electronic properties. Among various biomedical applications, CNTs also attract interest as nonviral gene delivery systems. Functionalization of CNTs with cationic groups enables delivery of negatively charged DNA into cells. In contrast to this well‐known strategy for DNA delivery, our approach included the covalent attachment of linearized plasmid DNA to carboxylated multiwalled CNTs (MWCNTs). Carboxyl groups were introduced onto MWCNTs by oxidative treatment, and then the carboxyl groups were activated by 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC). The whole pQE‐70 vector including the gene encoding green fluorescent protein (GFP) was subjected to polymerase chain reaction (PCR) using the modified nucleotide N6‐(6‐Amino)hexyl‐2′‐deoxyadenosine‐5′‐triphosphate. Hence, free amino groups were introduced onto the linearized plasmid. Covalent bonding between the amino‐modified plasmid DNA and the carboxylated MWCNTs was achieved via EDC chemistry. The resulting bioconjugate was successfully transformed into chemically competent Escherichia coli cells, without necessity of a heat‐shock step at 42°C. The presence of Ca²⁺ in transformation medium was required to neutralize the electrostatic repulsion between DNA and negatively charged outer layer of E. coli. The transformants, which were able to express GFP were inspected manually on ampicillin agar plates. Our study represents a novelty with respect to other noncovalent CNT gene delivery systems. Considering the interest for delivery of linear DNA fragments, our study could give insights into further studies. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:224–232, 2014     

Doxorubicin conjugated carbon dots as a drug delivery system for human breast cancer therapy

Objectives

Carbon dots (CDs) are one of the most promising carbon‐based materials in bioimaging and drug/gene delivery applications. In this study, we have attempted to study the drug carrying capacity of highly fluorescent CDs for delivery of doxorubicin (DOX) and investigate the therapeutic activity of the CDs‐DOX drug delivery system.

Materials and methods

Carbon dots were synthesized by means of a hydrothermal approach with mixing citric acid and ethylenediamine. The properties of CDs were characterized in respects of spectral property, zeta potential, particle morphology and chemical composition. The drug loading efficiency (DLE) and release profile of CDs‐DOX were determined by a fluorescence spectrophotometer. We investigated the cellular toxicity and pharmaceutical activity of CDs and CDs‐DOX in L929 cells and MCF‐7 cells by the CCK‐8 assay. We also studied the cellular uptake of CDs‐DOX with the methods of confocal microscopy and flow cytometry. In addition, the effect of CDs‐DOX on cell apoptosis was assessed by flow cytometry.

Results

The obtained CDs possessed good biocompatibility and showed a potential capacity of promoting proliferation. DOX was successfully conjugated to CDs through electrostatic interaction, and the results of the DLE and loading content (DLC) suggested a relatively high drug loading capacity of CDs. Compared with free DOX, the CDs‐DOX complex had a higher cellular uptake and better anti‐tumour efficacy on MCF‐7 cells.

Conclusions

The results of this study indicated that the CDs‐DOX drug delivery system had a potential value in cancer chemotherapeutic application.

     

Synthesis of PVA-CAP-based biomaterial in situ dispersed with Cu nanoparticles and carbon micro-nanofibers for antibiotic drug delivery applications

A novel pH-sensitive and water-soluble polyvinyl alcohol (PVA)-cellulose acetate phthalate (CAP) composite-based biomaterial was prepared, in which the multi-scale web of copper (Cu)-grown carbon micro-nanofibers (Cu-ACF/CNFs) was in situ dispersed during a synthesis stage. PVA-CAP and Cu-nanoparticles (NPs) were used as an encapsulating agent and nano-antibiotics, respectively. The web of Cu-ACF/CNF was prepared by growing CNFs on the activated carbon microfiber (ACF) substrate by chemical vapor deposition using Cu NPs as the catalyst. The novel step of the synthesis included esterification of polyvinyl acetate (PVAc) to produce a PVA gel to which the ball-milled Cu-ACF/CNF was blended at the incipience of the gel formation to produce the PVA-CAP-Cu-ACF/CNF metal-carbon-polymeric composite film. The in vitro dissolution tests revealed that the encapsulating polymeric composite was dispersible in water and its rate of dissolution was high at pH > 6.5. The antibacterial tests performed on the material demonstrated its effectiveness against both gram negative Escherichia coli and gram positive Staphylococcus aureus bacterial strains. The Hixson-Crowell kinetic model described the dissolution profiles of the material. The method of preparation is novel, simple, and environmentally friendly. The prepared biomaterial may be used in several biomedical applications, including wound healing and the controlled release of drugs in the antibiotic delivery system.     

Self-assembled virus-like particles from rotavirus structural protein VP6 for targeted drug delivery

Proteins of viral capsid may self-assemble into virus-like particles (VLPs) that can find many biomedical applications such as platform for drug delivery. In this paper, we describe preparation of VLPs by self-assembly of VP6, a rotavirus capsid protein that was chemically conjugated with doxorubicin (DOX), an anticancer drug. VP6 was first highly expressed in E. Coli, followed by purification and renaturation. DOX was then covalently attached to VP6 to form DOX-VP6 (DVP6) conjugates, which were subsequently self-assembled into VLPs under appropriate condition. Next, lactobionic acid (LA) was chemically linked to the surface of the VLPs. We demonstrated that the aforementioned nanosystem shows specific targeting to hepatoma cell line HepG2. The chemically functionalized VLPs, a kind of biological nanoparticles with excellent biocompatibility and biodegradability, can be prepared in large scale from E. Coli through our method, which may find practical applications in biomedicine.     

Chimeric ribonuclease as a source of human adapter protein for targeted drug delivery

Assembled modular complexes for targeted drug delivery can be based on strong non-covalent interactions between a cargo module containing an adapter protein and a docking tag fused to a targeting protein. We have recently constructed a completely humanized adapter/docking tag system based on interactions between 15 amino acid (Hu-tag) and 110 amino acid (HuS) fragments of human ribonuclease I (RNase I). Although recombinant HuS can be expressed and refolded into a functionally active form, the purification procedure is cumbersome and expensive, and more importantly, it yields a significant proportion of improperly folded proteins. Here we describe engineering, high-yield expression, and purification of a chimeric bovine/human RNase (BH-RNase) comprising 1-29 N-terminal amino acids of bovine ribonuclease A and 30-127 amino acids of human RNase I. Unlike RNase I, the chimeric BH-RNase can be cleaved by either subtilisin or proteinase K between A20 and S21, providing a functionally active HuS. The HuS obtained from chimeric BH-RNase differs from wild-type HuS by an N24T substitution; therefore, we have reverted this substitution by mutating N24 to T24 in BH-RNase. This BH-RNase mutant can also be cleaved by subtilisin or proteinase K yielding wild-type HuS. The affinity of HuS obtained from BH-RNase to Hu-tag is approximately five times higher than that for recombinant HuS, reflecting a higher percentage of properly folded proteins.     

Chitosan graft copolymer nanoparticles for oral protein drug delivery: preparation and characterization

Several novel functionalized graft copolymer nanoparticles consisting of chitosan (CS) and the monomer methyl methacrylate (MMA), N-dimethylaminoethyl methacrylate hydrochloride (DMAEMC), and N-trimethylaminoethyl methacrylate chloride (TMAEMC), which show a higher solubility than chitosan in a broader pH range, have been prepared by free radical polymerization. The nanoparticles were characterized in terms of particle size, zeta potential, TEM, and FT-IR. These nanoparticles were 150-280 nm in size and carried obvious positive surface charges. Protein-loaded nanoparticles were prepared, and their maximal encapsulation efficiency was up to 100%. In vitro release showed that these nanoparticles provided an initial burst release followed by a slowly sustained release for more than 24 h. These graft copolymer nanoparticles enhanced the absorption and improved the bioavailability of insulin via the gastrointestinal (GI) tract of normal male Sprague-Dawley (SD) strain rats to a greater extent than that of the phosphate buffer solution (PBS) of insulin.