Aptamer-displaying peptide amphiphile micelles as a cell-targeted delivery vehicle of peptide cargoes.

Peptide amphiphile micelles (PAMs) are attractive vehicles for the delivery of a variety of therapeutic and prophylactic peptides. However, a key limitation of PAMs is their lack of preferential targeting ability. In this paper, we describe our design of a PAM system that incorporates a DNA oligonucleotide amphiphile (antitail amphiphile-AA) to form A/PAMs. A cell-targeting DNA aptamer with a 3' extension sequence (tail) complementary to the AA is annealed to the surface to form aptamer-displaying PAMs (Aptamer~A/PAMs). Aptamer~A/PAMs are small, anionic, stable nanoparticles capable of delivering a large mass percentage peptide amphiphile (PA) compared to targeting DNA components. Aptamer~A/PAMs are stable for over 4 h in the presence of biological fluids. Additionally, the aptamer retains its cell-targeting properties when annealed to the A/PAM, thus leading to enhanced delivery to a specifically-targeted B-cell leukemia cell line. This exciting modular technology can be readily used with a library of different targeting aptamers and PAs, capable of improving the bioavailability and potency of the peptide cargo.

Osteotropic nanoscale drug delivery systems based on small molecule bone-targeting moieties.

Bone-targeted drug delivery is an active research area because successful clinical applications of this technology can significantly advance the treatment of bone injuries and disorders. Molecules with bone-targeting potential have been actively investigated as promising moieties in targeted drug delivery systems. In general, bone-targeting molecules are characterized by their high affinity for bone and their predisposition to persist in bone tissue for prolonged periods, while maintaining low systemic concentrations. Proteins, such as monoclonal antibodies, have shown promise as bone-targeting molecules; however, they suffer from several limitations including large molecular size, high production cost, and undesirable immune responses. A viable alternative associated with significantly less side effects is the use of small molecule-based targeting moieties. This review provides a summary of recent findings regarding small molecule compounds with bone-targeting capacity, as well as nanoscale targeted drug delivery approaches employing these molecules.

Adjuvant Delivery Method and Nanoparticle Charge Influence Peptide Amphiphile Micelle Vaccine Bioactivity.

Vaccines are an indispensable public health measure that have enabled the eradication, near elimination, and prevention of a variety of pathogens. As research continues and our understanding of immunization strategies develops, subunit vaccines have emerged as exciting alternatives to existing whole vaccine approaches. Unfortunately, subunit vaccines often possess weak antigenicity, requiring delivery devices and adjuvant supplementation to improve their utility. Peptide amphiphile micelles have recently been shown to function as both delivery devices and self-adjuvanting systems that can be readily associated with molecular adjuvants to further improve vaccine-mediated host immunity. While promising, many "design rules" associated with the plethora of underlying adjustable parameters in the generation of a peptide amphiphile micelle vaccine have yet to be uncovered. This work explores the impact micellar adjuvant complexation method and incorporated antigen type have on their ability to activate dendritic cells and induce antigen specific responses. Interestingly, electrostatic complexation of CpG to micelles resulted in improved in vitro dendritic cell activation over hydrophobic association and antigen|adjuvant co-localization influenced cell-mediated, but not antibody-mediated immune responses. These exciting results complement those previously published to build the framework of a micelle vaccine toolbox that can be leveraged for future disease-specific formulations.

Hydrogen Sulfide Delivery to Enhance Bone Tissue Engineering Cell Survival.

Though crucial for natural bone healing, local calcium ion (Ca2+) and phosphate ion (Pi) concentrations can exceed the cytotoxic limit leading to mitochondrial overload, oxidative stress, and cell death. For bone tissue engineering applications, H2S can be employed as a cytoprotective molecule to enhance mesenchymal stem cell (MSC) tolerance to cytotoxic Ca2+/Pi concentrations. Varied concentrations of sodium hydrogen sulfide (NaSH), a fast-releasing H2S donor, were applied to assess the influence of H2S on MSC proliferation. The results suggested a toxicity limit of 4 mM for NaSH and that 1 mM of NaSH could improve cell proliferation and differentiation in the presence of cytotoxic levels of Ca2+ (32 mM) and/or Pi (16 mM). To controllably deliver H2S over time, a novel donor molecule (thioglutamic acid-GluSH) was synthesized and evaluated for its H2S release profile. Excitingly, GluSH successfully maintained cytoprotective level of H2S over 7 days. Furthermore, MSCs exposed to cytotoxic Ca2+/Pi concentrations in the presence of GluSH were able to thrive and differentiate into osteoblasts. These findings suggest that the incorporation of a sustained H2S donor such as GluSH into CaP-based bone graft substitutes can facilitate considerable cytoprotection, making it an attractive option for complex bone regenerative engineering applications.

Nano-ceramic composite scaffolds for bioreactor-based bone engineering.

BACKGROUND: Composites of biodegradable polymers and bioactive ceramics are candidates for tissue-engineered scaffolds that closely match the properties of bone. We previously developed a porous, three-dimensional poly (D,L-lactide-co-glycolide) (PLAGA)/nanohydroxyapatite (n-HA) scaffold as a potential bone tissue engineering matrix suitable for high-aspect ratio vessel (HARV) bioreactor applications. However, the physical and cellular properties of this scaffold are unknown. The present study aims to evaluate the effect of n-HA in modulating PLAGA scaffold properties and human mesenchymal stem cell (HMSC) responses in a HARV bioreactor. QUESTIONS/PURPOSES: By comparing PLAGA/n-HA and PLAGA scaffolds, we asked whether incorporation of n-HA (1) accelerates scaffold degradation and compromises mechanical integrity; (2) promotes HMSC proliferation and differentiation; and (3) enhances HMSC mineralization when cultured in HARV bioreactors. METHODS: PLAGA/n-HA scaffolds (total number = 48) were loaded into HARV bioreactors for 6 weeks and monitored for mass, molecular weight, mechanical, and morphological changes. HMSCs were seeded on PLAGA/n-HA scaffolds (total number = 38) and cultured in HARV bioreactors for 28 days. Cell migration, proliferation, osteogenic differentiation, and mineralization were characterized at four selected time points. The same amount of PLAGA scaffolds were used as controls. RESULTS: The incorporation of n-HA did not alter the scaffold degradation pattern. PLAGA/n-HA scaffolds maintained their mechanical integrity throughout the 6 weeks in the dynamic culture environment. HMSCs seeded on PLAGA/n-HA scaffolds showed elevated proliferation, expression of osteogenic phenotypic markers, and mineral deposition as compared with cells seeded on PLAGA scaffolds. HMSCs migrated into the scaffold center with nearly uniform cell and extracellular matrix distribution in the scaffold interior. CONCLUSIONS: The combination of PLAGA/n-HA scaffolds with HMSCs in HARV bioreactors may allow for the generation of engineered bone tissue. CLINICAL RELEVANCE: In cases of large bone voids (such as bone cancer), tissue-engineered constructs may provide alternatives to traditional bone grafts by culturing patients' own MSCs with PLAGA/n-HA scaffolds in a HARV culture system.

Vasoactive Intestinal Peptide Amphiphile Micelle Chemical Structure and Hydrophobic Domain Influence Immunomodulatory Potentiation.

Vasoactive intestinal peptide (VIP) is a neuropeptide capable of downregulating innate immune responses in antigen presenting cells (APCs) by suppressing their pro-inflammatory cytokine secretion and cell surface marker expression. Though VIP's bioactivity could possibly be leveraged as a treatment for transplant tolerance, drug delivery innovation is required to overcome its intrinsically limited cellular delivery capacity. One option is to employ peptide amphiphiles (PAs) which are lipidated peptides capable of self-assembling into micelles in water that can enhance cellular association. With this approach in mind, a series of triblock VIP amphiphiles (VIPAs) has been synthesized to explore the influence of block arrangement and hydrophobicity on micelle biocompatibility and bioactivity. VIPA formulation has been found to influence the shape, size, and surface charge of VIPA micelles (VIPAMs) as well as their cytotoxicity and immunomodulatory effects. Specifically, the enclosed work provides strong evidence that cylindrical VIPAMs with aspect ratios of 1.5-150 and moderate positive surface charge are able to potentiate the bioactivity of VIP limiting TNF-α secretion and MHC II and CD86 surface expression on APCs. With these criteria, we have identified PalmK-(EK)4-VIP as our lead formulation, which showed comparable or enhanced anti-inflammatory effects relative to the unmodified VIP at all dosages evaluated. Additionally, the relationships between peptide block location and lipid block size provide further information on the chemical structure-function relationships of PA micelles for the delivery of VIP as well as potentially for other peptides more broadly.

4D Printing of shape-memory polymeric scaffolds for adaptive biomedical implantation.

4D printing has shown great potential in a variety of biomedical applications due to the adaptability and minimal invasiveness of fabricated devices. However, commonly employed shape memory polymers (SMPs) possess undesirable transition temperatures (Ttranss), leading to complications in implantation operations. Herein, we demonstrate 4D printing of a new SMP named poly(glycerol dodecanoate) acrylate (PGDA) with a Ttrans in a range of 20 °C - 37 °C making it appropriate for shape programming at room temperature and then shape deployment within the human body. In addition, the material possesses suitable rheological properties to allow for the fabrication of a variety of delicate 3D structures such as "triangular star", "six-petal flower", "honeycomb", "tube", tilted "truncated hollow cones", as well as overhanging "bridge", "cage", and "mesh". The printed 3D structures show shape memory properties including a large fixity ratio of 100% at 20 °C, a large recovery ratio of 98% at 37 °C, a stable cyclability of > 100 times, and a fast recovery speed of 0.4 s at 37 °C. Moreover, the Young's moduli of the printed structures can be decreased by 5 times due to the phase transition of PGDA, which is compatible with biological tissues. Finally, in vitro stenting and in vivo vascular grafting demonstrated the geometrical and mechanical adaptivity of the printed constructs for biomedical implantation. This newly developed PGDA SMP based 4D printing technology has the potential to pave a new route to the fabrication of shape memory scaffolds for personalized biomedical applications.

Effect of Dibasic Calcium Phosphate Incorporation on Cellulose Nanocrystal/Chitosan Hydrogel Properties for the Treatment of Vertebral Compression Fractures.

Vertebral compression fractures account for approximately 700,000 out of the 1.5 million total osteoporotic fractures that occur annually in the USA. There is growing interest in substituting currently utilized clinical treatments for vertebral compression fractures with an injectable, degradable, and bioactive system. In this research we studied the osteoinductive effect of calcium phosphate incorporation into cellulose nanocrystal/chitosan hydrogels with varying ratios of carbonate as an ionic crosslinker and genipin as a covalent crosslinker. As calcium and phosphate ions have been shown to be osteoinductive in time and concentration dependent manners, dibasic calcium phosphate was chosen as a bioactive additive due to its desirable controlled ion delivery potential. Gelation time, swelling ratio, erosion, compressive strength, and ion release behavior of different dibasic calcium phosphate incorporated hydrogels were evaluated. Mesenchymal stem cells were then exposed to mechanically competent hydrogels found capable of maintaining calcium and phosphate concentrations within the established bioactive range in order to assess their cytotoxicity and osteoinductivity. Our results demonstrate that hydrogels with higher covalent crosslinking possessed better mechanical properties and stabilities as well as more controlled calcium and phosphate ion release. Interestingly, dibasic calcium phosphate incorporation not only improved hydrogel bioactivity but also resulted in greater compressive strength.

Inductive co-crosslinking of cellulose nanocrystal/chitosan hydrogels for the treatment of vertebral compression fractures.

Vertebral compression fractures are a very common consequence of osteoporosis for which injection of a non-biodegradable, non-bioactive, mechanically-stiff polymer bone cement into the vertebral body is the most common treatment. Recently, there has been growing interest in using bioactive, degradable, and bone biomechanics-matching products as an alternative approach for treating these fractures. In this research, we focused on creating injectable, chitosan-based hydrogels that can convey mechanical strength similar to vertebral bone as well as possess inherent osteoinductivity. First, we investigated the effects of three different factors - 1) bioactive phosphate ionic crosslinking; 2) genipin covalent crosslinking; 3) mechanically reinforcing cellulose nanocrystal incorporation - on the material properties of chitosan-based hydrogels. Mesenchymal stem cells were then exposed to hydrogels with optimum mechanical properties and stability in order to assess the biological effects of the bioactive phosphate ionic crosslinker. Our results show that hydrogels with higher ionic and covalent crosslinking ratios supplemented with neutral cellulose nanocrystals possessed desirable compressive strength and stability. Also, the significant osteoinductivity of these composite hydrogels demonstrated their potential to function as an injectable system for the future treatment of vertebral compression fractures.

Immunomodulatory vasoactive intestinal peptide amphiphile micelles.

Two different vasoactive intestinal peptide (VIP) amphiphiles have been formulated which readily form micelles of varying shapes. Interestingly, VIP micelle structure has been found to directly correlate to anti-inflammatory behavior providing evidence that these biomaterials can serve as a promising new therapeutic modality.

Instructive Design of Triblock Peptide Amphiphiles for Structurally Complex Micelle Fabrication.

Hydrophobically driven self-assembly is a well-understood principle that has been shown to facilitate micelle formation. Although quite useful, the library of structures accessible is limited to only a few simplistic geometric configurations that are suboptimal for complex applications. It is believed that other physical phenomena like hydrogen bonding and electrostatic interactions can be exploited to complement hydrophobic interactions allowing for the design of structurally complex, aggregated micelles. To test this theory, ABC triblock peptide amphiphiles comprising an application-specific peptide, a zwitterion-like peptide, and a hydrophobic lipid were synthesized for which block sequence modifications and order changes were used to investigate their impact on micelle formation. The results provide significant evidence that both hydrophobic and electrostatic driving forces influence the formation of complex micellar structures. Specifically, hydrophobic self-assembly facilitates individual micelle formation, whereas dipole electrostatic interactions govern the association of micelle units into complex architectures. Initial results indicate that there exists considerable flexibility in the choice of application-specific peptide allowing these structures to serve as a platform technology for a variety of fields.

Peptide Amphiphile Micelle Vaccine Size and Charge Influence the Host Antibody Response.

Vaccines are one of the best health care advances ever developed, having led to the eradication of smallpox and near eradication of polio and diphtheria. While tremendously successful, traditional vaccines (i.e., whole-killed or live-attenuated) have been associated with some undesirable side effects, including everything from mild injection site inflammation to the autoimmune disease Guillain-Barré syndrome. This has led recent research to focus on developing subunit vaccines (i.e., protein, peptide, or DNA vaccines) since they are inherently safer because they deliver only the bioactive components necessary (i.e., antigens) to produce a protective immune response against the pathogen of interest. However, a major challenge in developing subunit vaccines is overcoming numerous biological barriers to effectively deliver the antigen to the secondary lymphoid organs where adaptive immune responses are orchestrated. Peptide amphiphile micelles are a class of biomaterials that have been shown to possess potent self-adjuvanting vaccine properties, but their optimization capacity and underlying immunostimulatory mechanism are not well understood. The present work investigated the influence of micelle size and charge on the materials' bioactivity, including lymph node accumulation, cell uptake ability, and immunogenicity. The results generated provide considerable insight into how micelles exert their biological effects, yielding a micellar toolbox that can be exploited to either enhance or diminish host immune responses. This exciting development makes peptide amphiphile micelles an attractive candidate for both immune activation and suppression applications.

Calcium and phosphate ions as simple signaling molecules with versatile osteoinductivity.

Due to the continually increasing clinical need to heal large bone defects, synthetic bone graft substitutes have become ever more necessary with calcium phosphates (CaP) widely used due to their similarity to the mineral component of bone. In this research, different concentrations of calcium ions (Ca2+), phosphate ions (Pi), or their combination were provided to mesenchymal stem cells (MSCs) to evaluate their influence on proliferation and differentiation. The results suggest that 1-16 mM Ca2+ and 1-8 mM Pi is osteoinductive, but not cytotoxic. Furthermore, three distinct calcium phosphates (i.e. monobasic, dibasic, and hydroxyapatite) with different dissolution rates were investigated for their Ca2+ and Pi release. These biomaterials were then adjusted to release ion concentrations within the established therapeutics window for which MSC bioactivity was assessed. These findings suggest that CaP-based biomaterials can be leveraged to achieve Ca2+ and Pi dose-dependent osteoinduction for bone regenerative engineering applications.

Vaccine Adjuvant Incorporation Strategy Dictates Peptide Amphiphile Micelle Immunostimulatory Capacity.

Current vaccine research has shifted from traditional vaccines (i.e., whole-killed or live-attenuated) to subunit vaccines (i.e., protein, peptide, or DNA) as the latter is much safer due to delivering only the bioactive components necessary to produce a desirable immune response. Unfortunately, subunit vaccines are very weak immunogens requiring delivery vehicles and the addition of immunostimulatory molecules termed adjuvants to convey protective immunity. An interesting type of delivery vehicle is peptide amphiphile micelles (PAMs), unique biomaterials where the vaccine is part of the nanomaterial itself. Due to the modularity of PAMs, they can be readily modified to deliver both vaccine antigens and adjuvants within a singular construct. Through the co-delivery of a model antigenic epitope (Ovalbumin319-340-OVABT) and a known molecular adjuvant (e.g., 2,3-dipalmitoyl-S-glyceryl cysteine-Pam2C), greater insight into the mechanisms by which PAMs can exert immunostimulatory effects was gained. It was found that specific combinations of antigen and adjuvant can significantly alter vaccine immunogenicity both in vitro and in vivo. These results inform fundamental design rules that can be leveraged to fabricate optimal PAM-based vaccine formulations for future disease-specific applications. Graphical Abstract.

Porous polymeric platform repairs primates after spinal cord injury.

Amine-containing polymers immobilized on mesh and placed at trauma sites scavenge biomolecules that initiate a damaging immune response.

DAMPening the effects of trauma-induced inflammation.

Amine-containing polymers immobilized on mesh and placed at trauma sites scavenge biomolecules that initiate a damaging immune response.

Modular Peptide Amphiphile Micelles Improving an Antibody-Mediated Immune Response to Group A Streptococcus.

Inducing a strong and specific immune response is the hallmark of a successful vaccine. Nanoparticles have emerged as promising vaccine delivery devices to discover and elicit immune responses. Fine-tuning a nanoparticle vaccine to create an immune response with specific antibody and other cellular responses is influenced by many factors such as shape, size, and composition. Peptide amphiphile micelles are a unique biomaterials platform that can function as a modular vaccine delivery system, enabling control over many of these important factors and delivering payloads more efficiently to draining lymph nodes. In this study, the modular properties of peptide amphiphile micelles are utilized to improve an immune response against a Group A Streptococcus B cell antigen (J8). The hydrophobic/hydrophilic interface of peptide amphiphile micelles enabled the precise entrapment of amphiphilic adjuvants which were found to not alter micelle formation or shape. These heterogeneous micelles significantly enhanced murine antibody responses when compared to animals vaccinated with nonadjuvanted micelles or soluble J8 peptide supplemented with a classical adjuvant. The heterogeneous micelle induced antibodies also showed cross-reactivity with wild-type Group A Streptococcus providing evidence that micelle-induced immune responses are capable of identifying their intended pathogenic targets.

Controlled Ion Release from Novel Polyester/Ceramic Composites Enhances Osteoinductivity.

Due to the growing number of patients suffering from musculoskeletal defects and the limited supply of and sub-optimal outcomes associated with biological graft materials, novel biomaterials must be created that can function as graft substitutes. For bone regeneration, composite materials that mimic the organic and inorganic phases of natural bone can provide cues which expedite and enhance endogenous repair. Specifically, recent research has shown that calcium and phosphate ions are inherently osteoinductive, so controllably delivering their release holds significant promise for this field. In this study, unique aliphatic polyesters were synthesized and complexed with a rapidly decomposing ceramic (monobasic calcium phosphate, MCP) yielding novel polymer/ceramic composite biomaterials. It was discovered that the fast dissolution and rapid burst release of ions from MCP could be modulated depending on polymer length and chemistry. Also, controlled ion release was found to moderate solution pH associated with polyester degradation. When composite biomaterials were incubated with mesenchymal stems cells (MSCs) they were found to better facilitate osteogenic differentiation than the individual components as evidenced by increased alkaline phosphate expression and more rapid mineralization. These results indicate that controlling calcium and phosphate ion release via a polyester matrix is a promising approach for bone regenerative engineering.

Nanoparticles as synthetic vaccines.

As vaccines have transitioned from the use of whole pathogens to only the required antigenic epitopes, unwanted side effects have been decreased, but corresponding immune responses have been greatly diminished. To enhance immunogenicity, a variety of controlled release vehicles have been proposed as synthetic vaccines, but nanoparticles have emerged as particularly impressive systems due to many exciting publications. In specific, nanoparticles have been shown capable of not only desirable vaccine release, but can also be targeted to immune cells of interest, loaded with immunostimulatory substances termed adjuvants, or even induce desirable immune activating effects on their own. In the present review, recent advances in the utilization of inorganic, polymeric, and biomolecular nanoparticles as synthetic vaccines are discussed.

Biocompatibility and characterization of a peptide amphiphile hydrogel for applications in peripheral nerve regeneration.

Peripheral nerve injury is a debilitating condition for which new bioengineering solutions are needed. Autografting, the gold standard in treatment, involves sacrifice of a healthy nerve and results in loss of sensation or function at the donor site. One alternative solution to autografting is to use a nerve guide conduit designed to physically guide the nerve as it regenerates across the injury gap. Such conduits are effective for short gap injuries, but fail to surpass autografting in long gap injuries. One strategy to enhance regeneration inside conduits in long gap injuries is to fill the guide conduits with a hydrogel to mimic the native extracellular matrix found in peripheral nerves. In this work, a peptide amphiphile (PA)-based hydrogel was optimized for peripheral nerve repair. Hydrogels consisting of the PA C16GSH were compared with a commercially available collagen gel. Schwann cells, a cell type important in the peripheral nerve regenerative cascade, were able to spread, proliferate, and migrate better on C16GSH gels in vitro when compared with cells seeded on collagen gels. Moreover, C16GSH gels were implanted subcutaneously in a murine model and were found to be biocompatible, degrade over time, and support angiogenesis without causing inflammation or a foreign body immune response. Taken together, these results help optimize and instruct the development of a new synthetic hydrogel as a luminal filler for conduit-mediated peripheral nerve repair.