Characterization and Optimization of Injectable In Situ Crosslinked Chitosan-Genipin Hydrogels.

In recent years, there has been an increased interest in injectable, in situ crosslinking hydrogels due to their minimally invasive application and ability to conform to their environment. Current in situ crosslinking chitosan hydrogels are either mechanically robust with poor biocompatibility and limited biodegradation due to toxic crosslinking agents or the hydrogels are mechanically weak and undergo biodegradation too rapidly due to insufficient crosslinking. Herein, the authors developed and characterized a thermally-driven, injectable chitosan-genipin hydrogel capable of in situ crosslinking at 37 °C that is mechanically robust, biodegradable, and maintain high biocompatibility. The natural crosslinker genipin is utilized as a thermally-driven, non-toxic crosslinking agent. The chitosan-genipin hydrogel's crosslinking kinetics, injectability, viscoelasticity, swelling and pH response, and biocompatibility against human keratinocyte cells are characterized. The developed chitosan-genipin hydrogels are successfully crosslinked at 37 °C, demonstrating temperature sensitivity. The hydrogels maintained a high percentage of swelling over several weeks before degrading in biologically relevant environments, demonstrating mechanical stability while remaining biodegradable. Long-term cell viability studies demonstrated that chitosan-genipin hydrogels have excellent biocompatibility over 7 days, including during the hydrogel crosslinking phase. Overall, these findings support the development of an injectable, in situ crosslinking chitosan-genipin hydrogel for minimally invasive biomedical applications.

Influence of extracellular cues of hydrogel biomaterials on stem cell fate.

Tissue engineering is a multidisciplinary field that focuses on creating functional tissue through the combination of biomimetic scaffolds, a cell source, and biochemical/physiochemical cues. Stem cells are often used as the cell source due to their multipotent properties and autologous sourcing; however, the combination of physical and chemical cues that regulate their behavior creates challenges in reproducibly directing them to a specific fate. Hydrogel biomaterials are widely explored as tissue scaffolds due to their innate biomimetic properties and tailorability. For these constructs to be successful, properties such as surface chemistry and spatial configuration, stiffness, and degradability of the biomaterial used for the scaffold framework should be analogous to the natural environment of the tissue they are repairing/replacing. This is imperative, as cues from the surrounding extracellular matrix (ECM) influence stem cell behavior and direct cell differentiation to a specific lineage. Hydrogels offer great promise as tools to control stem cell fate, as researchers can modulate the degradation rates, mechanical properties, swelling behavior, and chemical properties of the biomaterial scaffold to mimic the instructive cues of the native ECM. Discussion of the advantages and challenges of utilizing hydrogel biomaterials as the basis of tissue scaffolds is reviewed herein, as well as specific examples of hydrogels in tissue engineering and advances in hydrogel research to achieve desired cell phenotypes.

Combination of soluble factors and biomaterial scaffolds enhance human adipose-derived stem/stromal cell myogenesis.

Volumetric muscle loss and muscle degeneration are conditions for which there are currently no effective treatment options. Human adipose stem cells (hASCs) offer promise in cell-based regenerative therapies to treat muscle damage due to their ability to self-renew and differentiate. However, in the absence of universal culture conditions that yield greater than 15% myogenic differentiation, the clinical potential of these cells is limited. Here we report on the evaluation of two different media recipes, three extracellular matrix (ECM) proteins, and a poly (ethylene glycol) (PEGDMA) hydrogel with a physiologically relevant elasticity to determine how the extracellular chemical and physical environment work together to enhance myogenic differentiation of hASCs. Our results identify a combination of unique biochemical and physical factors that promote myogenesis, laying the groundwork for creating a scaffold and culture medium that will effectively and efficiently direct myogenic differentiation of adult stem cells for clinical applications in the future.

Poly (ethylene glycol) hydrogel scaffolds with multiscale porosity for culture of human adipose-derived stem cells.

Three-dimensional (3 D) hydrogel scaffolds are an attractive option for tissue regeneration applications because they allow for cell migration, fluid exchange, and can be synthesized to closely mimic the physical properties of the extracellular matrix environment. The material properties of hydrogels play a vital role in cellular migration and differentiation. In light of this, in-depth understanding of material properties is required before such scaffolds can be used to study their influence on cells. Herein, various blends and thicknesses of poly (ethylene glycol) dimethacrylate (PEGDMA) hydrogels were synthesized, flash frozen, and dried by lyophilization to create scaffolds with multiscale porosity. Environmental scanning electron microscopy (ESEM) images demonstrated that lyophilization induced microporous voids in the PEGDMA hydrogels while swelling studies show the hydrogels retain their innate swelling properties. Change in pore size was observed between drying methods, polymer blend, and thickness when imaged in the hydrated state. Human adipose-derived stem cells (hASCs) were seeded on lyophilized and non-lyophilized hydrogels to determine if the scaffolds would support cell attachment and proliferation of a clinically relevant cell type. Cell attachment and morphology of the hASCs were evaluated using fluorescence imaging. Qualitative observations in cell attachment and morphology of hASCs on the surface of the different hydrogel spatial configurations indicate these multiscale porosity hydrogels create a suitable scaffold for hASC culture. These findings offer another factor of tunability in creating biomimetic hydrogels for various tissue engineering applications including tissue repair, regeneration, wound healing, and controlled release of growth factors.

Effects of post-processing methods on chitosan-genipin hydrogel properties.

Biopolymer based hydrogel materials are attractive options for a variety of medical applications, including drug delivery and tissue regeneration because of their innate biomimetic material properties. While biopolymers are typically selected for a specific application based off of their chemical properties; the overall material structure of the resulting hydrogel ultimately relates to its ability to function for its intended application. In view of this, it is imperative that the impact of commonly used drying procedures on hydrogel physical properties is well understood. Herein, the effects of post-synthesis drying techniques: air-drying and lyophilization, on genipin crosslinked chitosan hydrogel physical structure were studied. Chitosan-genipin hydrogels synthesized out of either 15 kDa MW or 50-190 kDa MW chitosan were either air-dried (AD), flash-frozen (FF) and then lyophilized, or step-down frozen (SD) and then lyophilized. Environmental scanning electron microscopy (ESEM) was employed to evaluate the resulting hydrogels physical structure as a function of chitosan molecular weight and drying condition. ESEM images revealed the presence of larger microscale pores within the SD samples compared to FF samples, but both treatments yielded the induction of micropore with sizes ranging between 9-400 µm in diameter into the hydrogels. Traditional hydrogel swelling studies were performed to assess the resulting hydrogels swelling profile as a function of chitosan molecular weight and drying treatment. Lyophilized hydrogels showed a five-fold increase in swelling ratio compared to AD hydrogels indicating a change in morphology due to the drying process. The results demonstrate that regardless of polymer molecular weight, post-processing technique had a strong correlation with hydrogel porosity.

Development of Responsive Chitosan-Genipin Hydrogels for the Treatment of Wounds.

Chronic wounds are characterized by an increased bacterial presence, alkaline pH, and excessive wound drainage. Hydrogel biomaterials composed of the carbohydrate polymer chitosan are advantageous for wound healing applications because of their innate antimicrobial and hemostatic properties. Here, genipin-cross-linked-chitosan hydrogels were synthesized and characterized, and their in vitro and in vivo performances were evaluated as a viable wound dressing. Characterization studies demonstrate that the developed chitosan-genipin hydrogels were able to neutralize an environmental pH, while averaging ∼230% aqueous solution uptake, demonstrating their use as a perfusive wound dressing. Bacterial activity studies demonstrate the hydrogels' ability to hinder Escherichia coli growth by ∼70%, while remaining biocompatible in vitro to fibroblast and keratinocyte cells. Furthermore, chitosan-genipin hydrogels promote an enhanced immune response and cellular proliferation in induced pressure wounds in mice. All together, these results reflect the potential of the developed hydrogels to be used as a proactive wound dressing.

Transport and delivery of interferon-α through epithelial tight junctions via pH-responsive poly(methacrylic acid-grafted-ethylene glycol) nanoparticles.

Whereas significant advancements have been made in our fundamental understanding of cancer, they have not yet translated into effective clinical cancer treatments. One of the areas that has the potential to improve the efficacy of cancer therapies is the development of novel drug delivery technologies. In particular, the design of pH-sensitive polymeric complexation hydrogels may allow for targeted oral delivery of a wide variety of chemotherapeutic drugs and proteins. In this work, poly(methacrylic acid-grafted-ethylene glycol) hydrogel nanoparticles were synthesised, characterised, and studied as matrix-type, diffusion-controlled, pH-responsive carriers to enable the oral delivery of the chemotherapeutic agent interferon alpha (IFN-α). The biophysical mechanisms controlling the transport of IFN-α were investigated using a Caco-2/HT29-MTX co-culture as a gastrointestinal (GI) tract model. The synthesised nanoparticles exhibited pH-responsive swelling behaviour and allowed the permeation of IFN-α through the tight junctions of the developed cellular GI epithelium model. These studies demonstrate the capabilities of these particles to contribute to the improved oral delivery of protein chemotherapeutics.

Poly (ethylene glycol) hydrogel elasticity influences human mesenchymal stem cell behavior.

Coordinated investigations into the interactions between biologically mimicking (biomimetic) material constructs and stem cells advance the potential for the regeneration and possible direct replacement of diseased cells and tissues. Any clinically relevant therapies will require the development and optimization of methods that mass produce fully functional cells and tissues. Despite advances in the design and synthesis of biomaterial scaffolds, one of the biggest obstacles facing tissue engineering is understanding how specific extracellular cues produced by biomaterial scaffolds influence the proliferation and differentiation of various cell sources. Matrix elasticity is one such tailorable property of synthetic scaffolds that is known to differ between tissues. Here, we investigate the interactions between an elastically tailorable polyethylene glycol (PEG)-based hydrogel platform and human bone marrow-derived mesenchymal stem cells (hMSCs). For these studies, two different hydrogel compositions with elastic moduli in the ranges of 50-60 kPa and 8-10 kPa were implemented. Our findings demonstrate that the different elasticities in this platform can produce changes in hMSC morphology and proliferation, indicating that the platform can be implemented to produce changes in hMSC behavior and cell state for a broad range of tissue engineering and regenerative applications. Furthermore, we show that the platform's different elasticities influence stem cell differentiation potential, particularly when promoting stem cell differentiation toward cell types from tissues with stiffer elasticity. These findings add to the evolving and expanding library of information on stem cell-biomaterial interactions and opens the door for continued exploration into PEG-based hydrogel scaffolds for tissue engineering and regenerative medicine applications.

Poly(ethylene glycol) Hydrogels with Tailorable Surface and Mechanical Properties for Tissue Engineering Applications.

Advanced cellular biomanufacturing requires the large-scale production of biocompatible materials that can be utilized in the study of cell-matrix interactions and directed stem cell differentiation as well as the generation of physiologically relevant tissues for therapeutic applications. Herein we describe the development of a hydrogel based platform with tailorable mechanical properties that supports the attachment and proliferation of both pluripotent and multipotent stem cells. The biomimetic hydrogel scaffold generated provides biocompatible compositions for generating various tissue-like elasticities for regenerative medicine applications and advanced biomanufacturing.

A review of current nanoparticle and targeting moieties for the delivery of cancer therapeutics.

The tumor microenvironment provides unique challenges for the delivery of chemotherapeutic agents in doses that are effective while ensuring minimal systemic toxicity. The primary limitation of current therapeutics is a lack of specificity in delivery, as they target healthy and cancerous cells alike. The development of nanoscale carriers capable of delivering cancer therapies has the potential to overcome both systemic and tumor barriers and provide specific, targeted delivery. This review seeks to provide an overview of available nanoscale drug carriers by exploring the wide variety of developed nanostructures and the most commonly used moieties for targeted delivery. Additionally, the use of nanoscale carriers will be motivated by examining tumor physiology and the specific barriers present within both the tumor microenvironment and systemic delivery.

Responsive theranostic systems: integration of diagnostic imaging agents and responsive controlled release drug delivery carriers.

For decades, researchers and medical professionals have aspired to develop mechanisms for noninvasive treatment and monitoring of pathological conditions within the human body. The emergence of nanotechnology has spawned new opportunities for novel drug delivery vehicles capable of concomitant detection, monitoring, and localized treatment of specific disease sites. In turn, researchers have endeavored to develop an imaging moiety that could be functionalized to seek out specific diseased conditions and could be monitored with conventional clinical imaging modalities. Such nanoscale detection systems have the potential to increase early detection of pathophysiological conditions because they can detect abnormal cells before they even develop into diseased tissue or tumors. Ideally, once the diseased cells are detected, clinicians would like to treat those cells simultaneously. This idea led to the concept of multifunctional carriers that could target, detect, and treat diseased cells. The term "theranostics" has been created to describe this promising area of research that focuses on the combination of diagnostic detection agents with therapeutic drug delivery carriers. Targeted theranostic nanocarriers offer an attractive improvement to disease treatment because of their ability to execute simultaneous functions at targeted diseased sites. Research efforts in the field of theranostics encompass a broad variety of drug delivery vehicles, imaging contrast agents, and targeting modalities for the development of an all-in-one, localized detection and treatment system. Nanotheranostic systems that utilize metallic or magnetic imaging nanoparticles can also be used as thermal therapeutic systems. This Account explores recent advances in the field of nanotheranostics and the various fundamental components of an effective theranostic carrier.

Advanced molecular design of biopolymers for transmucosal and intracellular delivery of chemotherapeutic agents and biological therapeutics.

Hydrogels have been instrumental in the development of polymeric systems for controlled release of therapeutic agents. These materials are attractive for transmucosal and intracellular drug delivery because of their facile synthesis, inherent biocompatibility, tunable physicochemical properties, and capacity to respond to various physiological stimuli. In this contribution, we outline a multifaceted hydrogel-based approach for expanding the range of therapeutics in oral formulations from classical small-molecule drugs to include proteins, chemotherapeutics, and nucleic acids. Through judicious material selection and careful design of copolymer composition and molecular architecture, we can engineer systems capable of responding to distinct physiological cues, with tunable physicochemical properties that are optimized to load, protect, and deliver valuable macromolecular payloads to their intended site of action. These hydrogel carriers, including complexation hydrogels, tethered hydrogels, interpenetrating networks, nanoscale hydrogels, and hydrogels with decorated structures are investigated for their ability to respond to changes in pH, to load and release insulin and fluorescein, and remain non-toxic to Caco-2 cells. Our results suggest these novel hydrogel networks have great potential for controlled delivery of proteins, chemotherapeutics, and nucleic acids.

Designer nanoparticles: incorporating size, shape and triggered release into nanoscale drug carriers.

IMPORTANCE OF THE FIELD: Although significant progress has been made in delivering therapeutic agents through micro and nanocarriers, precise control over in vivo biodistribution and disease-responsive drug release has been difficult to achieve. This is critical for the success of next generation drug delivery devices, as newer drugs, designed to interfere with cellular functions, must be efficiently and specifically delivered to diseased cells. The chief constraint in achieving this has been our limited repertoire of particle synthesis methods, especially at the nanoscale. Recent developments in generating shape-specific nanocarriers and the potential to combine stimuli-responsive release with nanoscale delivery devices show great promise in overcoming these limitations. AREAS COVERED IN THIS REVIEW: How recent advances in fabrication technology allow synthesis of highly monodisperse, stimuli-responsive, drug-carrying nanoparticles of precise geometries is discussed. How particle properties, specifically shape and stimuli responsiveness, affect biodistribution, cellular uptake and drug release is also reviewed. WHAT THE READER WILL GAIN: The reader is introduced to recent developments in intelligent drug nanocarriers and new nanofabrication approaches that can be combined with disease-responsive biomaterials. This will provide insight into the importance of controlling particle geometry and incorporating stimuli-responsive materials into drug delivery. TAKE HOME MESSAGE: The integration of responsive biomaterials into shape-specific nanocarriers is one of the most promising avenues towards the development of next generation, advanced drug delivery systems.

Micro- and nanotechnologies for intelligent and responsive biomaterial-based medical systems.

Advances in medical treatments of a wide variety of pathophysiological conditions require the development of better therapeutic agents, as well as a combination of the required therapeutic agents with device-integrated biomaterials that can serve as sensors and carriers. Combination of micro- and nano-fabricated systems with intelligent biomaterials that have the ability to sense and respond is a promising avenue for the development of better diagnostic and therapeutic medical systems. Micro- and nano-electromechanical systems (MEMs and NEMs) are now becoming a family of potentially powerful new technologies for drug delivery, diagnostic tools, and tissue engineering. Improvements in micro- and nano-fabrication technologies have enhanced the ability to create better performing therapeutic systems for numerous pathophysiological applications. More importantly, MEMS- and NEMS-based tissue regeneration scaffolds, biosensors, and drug delivery devices provide new opportunities to mimic the natural intelligence and response of biological systems.

Nanoimprint lithography based fabrication of shape-specific, enzymatically-triggered smart nanoparticles.

Our ability to precisely manipulate size, shape and composition of nanoscale carriers is essential for controlling their in-vivo transport, bio-distribution and drug release mechanism. Shape-specific, "smart" nanoparticles that deliver drugs or imaging agents to target tissues primarily in response to disease-specific or physiological signals could significantly improve therapeutic care of complex diseases. Current methods in nanoparticle synthesis do not allow such simultaneous control over particle size, shape and environmentally-triggered drug release, especially at the sub 100 nm range. We report here a high-throughput nanofabrication technique using synthetic and biological macromers (peptides) to produce highly monodisperse, enzymatically-triggered nanoparticles of precise sizes and shapes. Particles as small as 50 nm were fabricated on silicon wafers and harvested directly into aqueous buffers using a biocompatible, one-step release technique. We further demonstrate successful encapsulation and precisely controlled enzyme-triggered release of antibodies and nucleic acids from these nanoparticles, thus providing a potential means for disease-controlled delivery of biomolecules.