Biomimetic approaches to protein and gene delivery for tissue regeneration

Novel therapeutic strategies that promote wound healing seek to mimic the response of the body to wounding, to regenerate rather than repair injured tissues. Many synthetic or natural biomaterials have been developed for this purpose and are used to deliver wound therapeutics in a controlled manner that prevents unwanted and potentially harmful side-effects. Here, we review the natural and synthetic biomaterials that have been developed for protein and gene delivery to enhance tissue regeneration. Particular emphasis is placed on novel biomimetic materials that respond to environmental stimuli or release their cargo according to cellular demand. Engineering biomaterials to release therapeutic agents in response to physiologic signals mimics the natural healing process and can promote faster tissue regeneration and reduce scarring in severe acute or chronic wounds.     

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.     

Molecular vehicles for targeted drug delivery

Targeted drug delivery by cell-specific cytokines and antibodies promises greater drug efficacy and reduced side effects. We describe a novel strategy for assembly of drug delivery vehicles that does not require chemical modification of targeting proteins. The strategy relies on a noncovalent binding of standardized "payload" modules to targeting proteins expressed with a "docking" tag. The payload modules are constructed by linking drug carriers to an adapter protein capable of binding to a docking tag. Using fragments of bovine ribonuclease A as an adapter protein and a docking tag, we have constructed vascular endothelial growth factor (VEGF) based vehicles for gene delivery and for liposome delivery. Assembled vehicles displayed remarkable selectivity in drug delivery to cells overexpressing VEGF receptors. We expect that our strategy can be employed for targeted delivery of many therapeutic or imaging agents by different recombinant targeting proteins.     

Review physical and chemical aspects of stabilization of compounds in silk

The challenge of stabilization of small molecules and proteins has received considerable interest. The biological activity of small molecules can be lost as a consequence of chemical modifications, while protein activity may be lost due to chemical or structural degradation, such as a change in macromolecular conformation or aggregation. In these cases, stabilization is required to preserve therapeutic and bioactivity efficacy and safety. In addition to use in therapeutic applications, strategies to stabilize small molecules and proteins also have applications in industrial processes, diagnostics, and consumer products like food and cosmetics. Traditionally, therapeutic drug formulation efforts have focused on maintaining stability during product preparation and storage. However, with growing interest in the fields of encapsulation, tissue engineering, and controlled release drug delivery systems, new stabilization challenges are being addressed; the compounds or protein of interest must be stabilized during: (1) fabrication of the protein or small molecule-loaded carrier, (2) device storage, and (3) for the duration of intended release needs in vitro or in vivo. We review common mechanisms of compound degradation for small molecules and proteins during biomaterial preparation (including tissue engineering scaffolds and drug delivery systems), storage, and in vivo implantation. We also review the physical and chemical aspects of polymer-based stabilization approaches, with a particular focus on the stabilizing properties of silk fibroin biomaterials.     

Hydrophobically modified biomineralized polysaccharide alginate membrane for sustained smart drug delivery

Hydrophobically modified biomineralized polysaccharide alginate membrane with smart drug release property using sodium palmitate as the hydrophobic component was prepared via a one-step method. The formation of CaHPO(4) in the membrane was clearly identified through scanning electron microscopy (SEM), energy dispersive X-ray spectrometer (EDS), X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy. Indomethacin release profiles of the modified alginate membrane were found to be pH- and thermo-responsive. The drug release of modified alginate membrane was around 60% within 12 h, while that of the alginate membrane was higher than 90%. These results indicate that the hydrophobic and biomineralized polysaccharide components can hinder the permeation of the encapsulated drug and reduce the drug release effectively. The resulting membrane can be used as "smart" materials for sustained dual-responsive drug delivery.     

Neighboring group-controlled hydrolysis: towards "designer" drug release biomaterials

To give the first demonstration of neighboring group-controlled drug delivery rates, a series of novel, polymerizable ester drug conjugates was synthesized and fully characterized. The monomers are suitable for copolymerization in biomaterials where control of drug release rate is critical to prophylaxis or obviation of infection. The incorporation of neighboring group moieties differing in nucleophilicity, geometry, and steric bulk in the conjugates allowed the rate of ester hydrolysis, and hence drug liberation, to be rationally and widely controlled. Solutions (2.5 x 10-5 mol dm-3) of ester conjugates of nalidixic acid incorporating pyridyl, amino, and phenyl neighboring groups hydrolyzed according to first-order kinetics, with rate constants between 3.00 +/- 0.12 x 10-5 s -1 (fastest) and 4.50 +/- 0.31 x 10- 6 s-1 (slowest). The hydrolysis was characterized using UV-visible spectroscopy. When copolymerized with poly(methyl methacrylate), free drug was shown to elute from the resulting materials, with the rate of release being controlled by the nature of the conjugate, as in solution. The controlled molecular architecture demonstrated by this system offers an attractive class of drug conjugate for the delivery of drugs from polymeric biomaterials such as bone cements in terms of both sustained, prolonged drug release and minimization of mechanical compromise as a result of release. We consider these results to be the rationale for the development of "designer" drug release biomaterials, where the rate of required release can be controlled by predetermined molecular architecture.     

Advances in polymeric systems for tissue engineering and biomedical applications

The characteristics of tissue engineered scaffolds are major concerns in the quest to fabricate ideal scaffolds for tissue engineering applications. The polymer scaffolds employed for tissue engineering applications should possess multifunctional properties such as biocompatibility, biodegradability and favorable mechanical properties as it comes in direct contact with the body fluids in vivo. Additionally, the polymer system should also possess biomimetic architecture and should support stem cell adhesion, proliferation and differentiation. As the progress in polymer technology continues, polymeric biomaterials have taken characteristics more closely related to that desired for tissue engineering and clinical needs. Stimuli responsive polymers also termed as smart biomaterials respond to stimuli such as pH, temperature, enzyme, antigen, glucose and electrical stimuli that are inherently present in living systems. This review highlights the exciting advancements in these polymeric systems that relate to biological and tissue engineering applications. Additionally, several aspects of technology namely scaffold fabrication methods and surface modifications to confer biological functionality to the polymers have also been discussed. The ultimate objective is to emphasize on these underutilized adaptive behaviors of the polymers so that novel applications and new generations of smart polymeric materials can be realized for biomedical and tissue engineering applications.     

Tales of biomaterials, molecules, and cells for repairing and treating brain dysfunction

Current therapies have limited or no capacity to restore lost function, slow ongoing neurodegeneration, or promote regeneration following damage to the brain. Biomaterials are playing an increasingly important role in the development of novel, potentially efficacious approaches to brain treatment and repair. Programmable biomaterials enable and augment the targeted delivery of drugs into the brain and allow cell/tissue transplants to be effectively delivered and integrate into the brain, to serve as delivery vehicles for therapeutic proteins, and rebuild damaged circuits. Similarly, biomaterials are being increasingly used to recapitulate specific aspects of brain niches to promote regeneration and/or repair damaged neuronal pathways with stem cell therapies. Many of these approaches are gaining momentum because nanotechnology allows greater control over material-cell interactions that induce specific developmental processes and cellular responses including differentiation, migration, and outgrowth. This review discusses the state of the art and new directions in the convergence of biomaterial science, drug delivery, and stem cell biology in the treatment of degenerative and malignant brain diseases.     

Biophysical characterization of layer‐by‐layer synthesis of aptamer‐drug microparticles for enhanced cell targeting

Targeted delivery of drug molecules to specific cells in mammalian systems demonstrates a great potential to enhance the efficacy of current pharmaceutical therapies. Conventional strategies for pharmaceutical delivery are often associated with poor therapeutic indices and high systemic cytotoxicity, and this result in poor disease suppression, low surviving rates, and potential contraindication of drug formulation. The emergence of aptamers has elicited new research interests into enhanced targeted drug delivery due to their unique characteristics as targeting elements. Aptamers can be engineered to bind to their cognate cellular targets with high affinity and specificity, and this is important to navigate active drug molecules and deliver sufficient dosage to targeted malignant cells. However, the targeting performance of aptamers can be impacted by several factors including endonuclease‐mediated degradation, rapid renal filtration, biochemical complexation, and cell membrane electrostatic repulsion. This has subsequently led to the development of smart aptamer‐immobilized biopolymer systems as delivery vehicles for controlled and sustained drug release to specific cells at effective therapeutic dosage and minimal systemic cytotoxicity. This article reports the synthesis and in vitro characterization of a novel multi‐layer co‐polymeric targeted drug delivery system based on drug‐loaded PLGA‐Aptamer‐PEI (DPAP) formulation with a stage‐wise delivery mechanism. A thrombin‐specific DNA aptamer was used to develop the DPAP system while Bovine Serum Albumin (BSA) was used as a biopharmaceutical drug in the synthesis process by ultrasonication. Biophysical characterization of the DPAP system showed a spherical shaped particulate formulation with a unimodal particle size distribution of average size ～0.685 µm and a zeta potential of +0.82 mV. The DPAP formulation showed a high encapsulation efficiency of 89.4 ± 3.6%, a loading capacity of 17.89 ± 0.72 mg BSA protein/100 mg PLGA polymeric particles, low cytotoxicity and a controlled drug release characteristics in 43 days. The results demonstrate a great promise in the development of DPAP formulation for enhanced in vivo cell targeting. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:249–261, 2018     

Drug delivery to target the posterior segment of the eye

Retinal diseases are nowadays the most common causes of vision threatening in developed countries. Therapeutic advances in this field are hindered by the difficulty to deliver drugs to the posterior segment of the eye. Due to anatomical barriers, the ocular biodisponibility of systemically administered drugs remains poor, and topical instillation is not adequate to achieve therapeutic concentrations of drugs in the back of the eye. Ocular drug delivery has thus become one of the main challenges of modern ophthalmology. A multidisciplinary research is being conducted worldwide including pharmacology, biomaterials, ophthalmology, pharmaceutics, and biology. New promising fields have been developed such as implantable or injectable slow release intravitreal devices and degradable polymers, dispersed polymeric systems for intraocular drug delivery, and transscleral delivery devices such as iontophoresis, osmotic pumps or intra-scleraly implantable materials. The first clinical applications emerging from this research are now taking place, opening new avenues for the treatment of retinal diseases.     

In silico Structure Modeling and Comparative Analysis of Characterization Properties of Protein Polymers Useful for Protein-Based Nano Particulate Drug Delivery Systems (NPDDS): A Bioinformatics Approach

With an increasing interest in nanoparticulate delivery systems, there is a greater need to identify biomaterials that are biocompatible and safe for human applications. Protein polymers from animal and plant sources are promising materials for designing nanocarriers. Composition of the protein plays an important role for specific drug delivery applications such as drug release, targeting, and stimuli responsive drug release. An important issue in protein polymers is characteristics such as size, charge, and hydrophobicity may play a significant role in phagocytic uptake and initiating a subsequent immune response. This remains to be investigated systematically by analyzing factors that influence nanoparticle characteristics of protein and reduce phagocytic uptake and does not initiate immune response too. Although protein polymers are biodegradable, it is essential to ensure that there must not be premature enzymatic breakdown of the protein nanoparticles in the systemic circulation. Surface modification of the protein nanoparticles can be used to address this issue to propose the necessary modification in the surface of the protein would be great contribution in the nano particulate drug delivery systems (NPPDS). Of the various proteins, gelatin and albumin have been widely studied for drug delivery applications. Plant proteins are yet to be investigated widely for drug delivery applications so there is need to find out the plant proteins capable to act as nanoparticles. The commercial success of albumin-based nanoparticles has created an interest in other proteins. An increased understanding of the physicochemical properties coupled with the developments in rDNA technology will open up new opportunities for protein-based nanoparticulate systems. In the present studies several proteins currently useful for drug delivery system were structurally modeled and has been analyzed to propose the essential characteristics of protein for protein-based NPDDS.     

Development and therapeutic applications of advanced biomaterials

Millions of patients worldwide have benefited from technological innovation from biomaterials. Yet, while life expectancy continues to increase, organ failure and traumatic injury continue to fill hospitals and diminish the quality of life. Advances in understanding disease and tissue regeneration combined with increased accessibility of modern technology have created new opportunities for the use of biomaterials in unprecedented ways. Materials can now be rapidly created and selected to target specific cells, change shape in response to external stimulus, and instruct tissue regeneration. Here we describe a few of these technologies with emphasis on targeted drug delivery vehicles, high-throughput material synthesis, minimally invasive biodegradable shape-memory materials, and development of strategies to enhance tissue regeneration through delivery of instructive materials.     

载基因洗脱支架防治再狭窄的研究进展

药物洗脱支架(DES)在冠状动脉疾病的治疗中起到巨大作用,不但能机械支撑血管狭窄区,而且可以通过持续释放药物显著降低病灶处再狭窄率。然而,长期临床研究表明,载药DES在后期有引发血栓的风险。在DES表面载入基因药物,通过表面涂层输送系统局部缓慢释放治疗基因,能针对引起再狭窄的细胞过程进行修改。选择合适的治疗基因,可以抑制内膜增生,促进再内皮化,提高洗脱支架的有效性和安全性,是非常有前途的抗再狭窄方法。同时,良好的涂层材料不仅改善了支架表面的生物相容性,更能通过不同的基因药物输送系统有效控制治疗基因的释放速率。本文首先介绍了一部分针对再狭窄的治疗基因,在此基础上,综合阐述了基因缓释系统中使用的材料和技术,分析提炼了基因缓释系统的释放机理,举例分析了载基因洗脱支架的研究进展,并展望了该领域的发展前景。     

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.     

Atomistic modeling of water diffusion in hydrolytic biomaterials

One of the most promising applications of hydrolytically degrading biomaterials is their use as drug release carriers. These uses, however, require that the degradation and diffusion of drug are reliably predicted, which is complex to achieve through present experimental methods. Atomistic modeling can help in the knowledge-based design of degrading biomaterials with tuned drug delivery properties, giving insights on the small molecules diffusivity at intermediate states of the degradation process. We present here an atomistic-based approach to investigate the diffusion of water (through which hydrolytic degradation occurs) in degrading bulk models of poly(lactic acid) or PLA. We determine the water diffusion coefficient for different swelling states of the polymeric matrix (from almost dry to pure water) and for different degrees of degradation. We show that water diffusivity is highly influenced by the swelling degree, while little or not influenced by the degradation state. This approach, giving water diffusivity for different states of the matrix, can be combined with diffusion-reaction analytical methods in order to predict the degradation path on longer time scales. Furthermore, atomistic approach can be used to investigate diffusion of other relevant small molecules, eventually leading to the a priori knowledge of degradable biomaterials transport properties, helping the design of the drug delivery systems.     

Injectable Lipid Emulsions—Advancements,Opportunities and Challenges

Injectable lipid emulsions, for decades, have been clinically used as an energy source for hospitalized patients by providing essential fatty acids and vitamins. Recent interest in utilizing lipid emulsions for delivering lipid soluble therapeutic agents, intravenously, has been continuously growing due to the biocompatible nature of the lipid-based delivery systems. Advancements in the area of novel lipids (olive oil and fish oil) have opened a new area for future clinical application of lipid-based injectable delivery systems that may provide a better safety profile over traditionally used long- and medium-chain triglycerides to critically ill patients. Formulation components and process parameters play critical role in the success of lipid injectable emulsions as drug delivery vehicles and hence need to be well integrated in the formulation development strategies. Physico-chemical properties of active therapeutic agents significantly impact pharmacokinetics and tissue disposition following intravenous administration of drug-containing lipid emulsion and hence need special attention while selecting such delivery vehicles. In summary, this review provides a broad overview of recent advancements in the field of novel lipids, opportunities for intravenous drug delivery, and challenges associated with injectable lipid emulsions.     

Ligand-decorated nanogels: fast one-pot synthesis and cellular targeting

Nanoscale vehicles for delivery have been of interest and extensively studied for two decades. However, the encapsulation stability of hydrophobic drug molecules in delivery vehicles and selective targeting these vehicles into disease cells are potential hurdles for efficient delivery systems. Here we demonstrate a simple and fast synthetic protocol of nanogels that shows high encapsulation stabilities. These nanogels can also be modified with various targeting ligands for active targeting. We show that the targeting nanogels (T-NGs), which are prepared within 2 h by a one-pot synthesis, exhibit very narrow size distributions and have the versatility of surface modification with cysteine-modified ligands including folic acid, cyclic arginine-glycine-aspartic acid (cRGD) peptide, and cell-penetrating peptide. T-NGs hold their payloads, undergo facilitated cell internalization by receptor-mediated uptake, and release their drug content inside cells due to the reducing intracellular environment. Selective cytotoxicity to cells, which have complementary receptors, is also demonstrated.     

Structural modification of protease inducible preprogrammed nanofiber precursor

As many proteases such as urokinase plasminogen activator (uPA) are overexpressed in various tumors, a new type of peptide-based smart delivery system (hydrogel matrix) that could be degraded by uPA was previously described (Law, B.; Weissleder, R.; Tung, C. H. Peptide-based biomaterials for protease-enhanced drug delivery. Biomacromolecules 2006, 7 (4), 1261-1265). Subsequently, we designed nanometer-sized fluorescent nanofibers by introducing a hydrophilic component (methoxyl polyethylene glycol) to the core peptide [MPEG 2000-BK(FITC)SGRSANA-kldlkldlkldl-NH 2]. Preliminary studies showed that these nanofibers could detect uPA activity by optical imaging in vitro (Law, B.; Weissleder, R.; Tung, C. H. Protease-sensitive fluorescent nanofibers. Bioconjugate Chem. 2007, 18 (6), 1701-1704). Here, we further extend our studies to the structural responses of these nanofiber precursors (NFP). In the presence of a model protease, the FITC-containing hydrophilic fragments were released from the NFPs that contributed to fluorescence amplification. Simultaneously, the remaining self-assembling residues were mechanically driven to transform into interfibril networking of micrometer size hydrogel. These unique morphological changes, together with the optical property, may have considerable biomedical applications as diagnostic sensors for specific protease or dual systemic and functional delivery nanoplatforms to target protease-associated diseases.     

A modular approach to the design of protein-based smart gels

The modular nature of repeat proteins makes them a versatile platform for the design of smart materials with predetermined properties. Here, we present a general strategy for combining protein modules with specified stability and function into arrays for the assembly of stimuli-responsive gels. We have designed tetratricopeptide repeat (TPR) arrays which contain peptide-binding modules that specify the strength and reversibility of network crosslinking in combination with spacer modules that specify crosslinking geometry and overall stability of the array. By combining such arrays with multivalent peptide ligands, self-supporting stimuli-responsive gels are formed. Using microrheology, we characterized the kinetics of gelation as a function of concentration and stoichiometry of the components. We also show that such gels are effective in encapsulating and releasing small molecules. Moreover, TPR gels alone are fully compatible with cell growth, whereas gels loaded with an anticancer compound release the compound, resulting in cell death. Thus, we have demonstrated that this new class of tunable biomaterials is ripe for further development as tissue engineering and drug delivery platform.     

Tunable self-assembly of genetically engineered silk--elastin-like protein polymers

Silk--elastin-like protein polymers (SELPs), consisting of the repeating units of silk and elastin blocks, combine a set of outstanding physical and biological properties of silk and elastin. Because of the unique properties, SELPs have been widely fabricated into various materials for the applications in drug delivery and tissue engineering. However, little is known about the fundamental self-assembly characteristics of these remarkable polymers. Here we propose a two-step self-assembly process of SELPs in aqueous solution for the first time and report the importance of the ratio of silk-to-elastin blocks in a SELP's repeating unit on the assembly of the SELP. Through precise tuning of the ratio of silk to elastin, various structures including nanoparticles, hydrogels, and nanofibers could be generated either reversibly or irreversibly. This assembly process might provide opportunities to generate innovative smart materials for biosensors, tissue engineering, and drug delivery. Furthermore, the newly developed SELPs in this study may be potentially useful as biomaterials for controlled drug delivery and biomedical engineering.