Functionally graded hydrogels with opposing biochemical cues for osteochondral tissue engineering.

Osteochondral tissue (OC) repair remains a significant challenge in the field of musculoskeletal tissue engineering. OC tissue displays a gradient structure characterised by variations in both cell types and extracellular matrix components from cartilage to the subchondral bone. These functional gradients in the native tissue have been replicated to engineer osteochondral tissue in vitro. While diverse fabrication methods have been employed to create these microenvironments, emulating the natural gradients and effective regeneration of the tissue continues to present a significant challenge. In this study, we present the design and development of an interpenetrating (IPN) hydrogel with opposing dual biochemical gradients with the aim of regenerating the complete osteochondral unit. The gradients of biochemical cues were generated using an in-house built extrusion system in CMC-Silk IPN hydrogel having improved mechanical strength. Firstly, we fabricated a hydrogel that exhibits a smooth transition of sCMC and TGF-ß1 (SCT gradient hydrogel) from the upper to the lower region of the IPN hydrogel, with the intent of regenerating the cartilage layer. Secondly, a hydrogel with a HAp gradient (HAp gradient hydrogel) from the lower to the upper region was fabricated to facilitate the regeneration of the subchondral bone layer. Subsequently, we developed a dual biochemical gradient hydrogel with the goal of regenerating the entire osteochondral unit. The fabricated hydrogel demonstrated a smooth transition of sCMC + TGF-ß1 and HAp gradients in opposing directions, along with a blend of both the biochemical cues at the interface. The results showed that the hydrogels having biochemical cues corresponding to the three zones (i.e., cartilage, interface and bone) of dual biochemical gradient hydrogel led to differentiation of BMSCs towards their respective lineages, thereby demonstrating their efficacy in directing the fate of progenitor cells. The developed dual gradient hydrogel provided biochemical signals that have the potential to facilitate tissue growth and regeneration of the entire osteochondral tissue with a smooth transition from cartilage (soft) to bone (hard) tissues. In summary, our study provides a simple and innovative method for incorporating biochemical cues into hydrogels. This promising approach is translatable and has the potential to be extrapolated to other interface tissues. .

Polyhydroxybutyrate-based osteoinductive mineralized electrospun structures that mimic components and tissue interfaces of the osteon for bone tissue engineering.

Scaffolds for bone tissue engineering should enable regeneration of bone tissues with its native hierarchically organized extracellular matrix (ECM) and multiple tissue interfaces. To achieve this, inspired by the structure and properties of bone osteon, we fabricated polyhydroxybutyrate (PHB)-based mineralized electrospun fibrous scaffolds. After studying multiple PHB-based fibers, we chose 7%PHB/1%Gelatin fibers (PG) to fabricate mineralized fibers that mimic mineralized collagen fibers in bone. The mineralized PG (mPG) surface had a rough, hydrophilic layer of low crystalline calcium phosphate which was biocompatible to bone marrow stromal cells (BMSCs), induced their proliferation and was osteoinductive. Subsequently, by modulating the electrospinning process, we fabricated mPG-based novel higher order fibrous scaffolds that mimic the macroscale geometries of osteons of bone ECM. Inspired by the aligned collagen fibers in bone lamellae, we fabricated mPG scaffolds with aligned fibers that could direct anisotropic elongation of mouse BMSC (mBMSCs). Further, we fabricated electrospun mPG-based osteoinductive tubular constructs which can mimic cylindrical bone components like osteons or lamellae or be used as long bone analogues based on their dimensions. Finally, to regenerate tissue interfaces in bone, we introduced a novel bi-layered scaffold-based approach. An electrospun bi-layered tubular construct that had PG in the outer layer and 7%PHB/0.5%Polypyrrole fibers (PPy) in the inner layer was fabricated. The bi-layered tubular construct underwent preferential surface mineralization only on its outer layer. This outer mineralized layer supported osteogenesis while the inner PPy layer could support neural cell growth. Thus, the bi-layered tubular construct may be used to regenerate haversian canal in the osteons which hosts nerve fibers. Overall, the study introduced novel techniques to fabricate biomimetic structures that can regenerate components of bone osteon and its multiple tissue interfaces. The study lays foundation for the fabrication of a modular scaffold that can regenerate bone with its hierarchical structure and complex tissue interfaces.

Engineering sulfated polysaccharides and silk fibroin based injectable IPN hydrogels with stiffening and growth factor presentation abilities for cartilage tissue engineering.

The extracellular matrix (ECM) presents a framework for various biological cues and regulates homeostasis during both developing and mature stages of tissues. During development of cartilage, the ECM plays a critical role in endowing both biophysical and biochemical cues to the progenitor cells. Hence, designing microenvironments that recapitulate these biological cues as provided by the ECM during development may facilitate the engineering of cartilage tissue. In the present study, we fabricated an injectable interpenetrating hydrogel (IPN) system which serves as an artificial ECM and provides chondro-inductive niches for the differentiation of stem cells to chondrocytes. The hydrogel was designed to replicate the gradual stiffening (as a biophysical cue) and the presentation of growth factors (as a biochemical cue) as provided by the natural ECM of the tissue, thus exemplifying a biomimetic approach. This dynamic stiffening was achieved by incorporating silk fibroin, while the growth factor presentation was accomplished using sulfated-carboxymethyl cellulose. Silk fibroin and sulfated-carboxymethyl cellulose (s-CMC) were combined with tyraminated-carboxymethyl cellulose (t-CMC) and crosslinked using HRP/H2O2 to fabricate s-CMC/t-CMC/silk IPN hydrogels. Initially, the fabricated hydrogel imparted a soft microenvironment to promote chondrogenic differentiation, and with time it gradually stiffened to offer mechanical support to the joint. Additionally, the presence of s-CMC conferred the hydrogel with the property of sequestering cationic growth factors such as TGF-ß and allowing their prolonged presentation to the cells. More importantly, TGF-ß loaded in the developed hydrogel system remained active and induced chondrogenic differentiation of stem cells, resulting in the deposition of cartilage ECM components which was comparable to the hydrogels that were treated with TGF-ß provided through media. Overall, the developed hydrogel system acts as a reservoir of the necessary biological cues for cartilage regeneration and simultaneously provides mechanical support for load-bearing tissues such as cartilage.

Directing ligament-mimetic bi-directional cell organization in scaffolds through zone-specific microarchitecture for ligament tissue engineering.

Ligament tissues exhibit zone-specific anisotropic cell organization. The cells in ligament-proper are longitudinally oriented, whereas, the cells in epiligament are circumferentially oriented. Therefore, scaffolds developed to regenerate ligament tissues should possess adequate architectural features to govern ligament-mimetic bi-directional cell organization. The scaffold architectural features along with ligament-mimetic cell organization may ultimately yield neo-tissues with ligament-like extracellular matrix (ECM) structure and biomechanical properties. Towards this goal, we fabricated a silk/gelatin-based core-shell scaffold (csSG) with zone-specific anisotropic architectural features, wherein, the core of the scaffold possessed longitudinally aligned pores while the shell of the scaffold possessed parallel microgrooves that are aligned circumferentially around the surface of the scaffold. The ligament-mimetic architectural features significantly improved the mechanical properties of the scaffold. Moreover, architectural features of the csSG scaffold governed zone-specific anisotropic organization of cells. The cells in the core were longitudinally oriented as observed in the ligament-proper and the cells on the shell were circumferentially oriented as observed in epiligament. This bi-directional cell orientation partially mimicked the complex cellular network in native ligament tissue. Additionally, both the core and the shell individually supported fibrogenic differentiation of stem cells which further improved their potential for ligament tissue engineering. Further, the aligned pores of the core could govern unidirectional organization of ECM deposited by cells which is crucial for regenerating anisotropic tissues like ligaments. Finally, when implanted subcutaneously in mice, the scaffolds retained their anisotropic architecture for at least 2 weeks, were biocompatible, supported cell infiltration and governed anisotropic organization of cells and ECM. Taken together, the fabricated biomimetic csSG scaffold, through its zone-specific architectural features, could govern ligament-mimetic cellular and ECM organization which is ultimately expected to achieve regeneration of ligament tissues with native-like hierarchical structure and biomechanical properties. Consequently, this study introduces bi-directional structural parameters as design criteria for developing scaffolds for ligament tissue engineering.

Piperlongumine mediates amelioration of osteoarthritis via inhibition of chondrocyte senescence and inflammation in a goat ex vivo model.

In osteoarthritis (OA), chondrocytes manifest senescence, which results in a vicious signaling loop that aids the progression of the disease. More specifically, inflammation-associated senescence is one of the major regulators of the initiation and progression of OA. Therefore, we targeted senescence through inflammation with a pharmacological approach for OA amelioration. In this study, we first confirmed the suitability of the IL1ß-induced goat ex vivo OA model (emphasizing 3R's principle) for the screening of senotherapeutics, namely, ABT-263, ABT-737, and Piperlongumine (PL), wherein PL showed a positive outcome in the preliminary studies. Thereafter, we determined the cytocompatible concentrations of PL using live/dead staining. Further, treatment of ex vivo OA cartilage with PL exhibited a concentration-dependent increase in the retention of key cartilage matrix components. We then examined the effect of PL on chondrocyte senescence and observed a decreased expression of major senescence markers in the PL-treated groups. Interestingly, PL treatment reduced the expression of major downstream effectors of the chondrocyte senescence pathway in a concentration-dependent manner at both gene and protein levels. Moreover, IL1ß-induced elevated levels of oxidative stress and DNA damage in cartilage explants were rescued by all the tested concentrations of PL. In addition, PL also reduced the expression of major inflammatory markers of OA in the goat ex vivo OA model. Finally, we proposed a model for the mechanism of action of PL in the treatment of OA. Overall, PL showed a promising outcome as a senotherapeutic for the amelioration of OA in the goat ex vivo OA model.

Targeting multiple disease hallmarks using a synergistic disease-modifying drug combination ameliorates osteoarthritis via inhibition of senescence and inflammation.

AIMS: Osteoarthritis (OA), is a debilitating disease characterized by progressive cartilage degradation, synovial inflammation, and chondrocyte senescence. Various treatment agents independently targeting these hallmarks have been investigated. However, due to the complex multifaceted nature of OA, no disease-modifying osteoarthritis drugs are clinically available. In an attempt to overcome this, we developed a combinatorial approach and demonstrated the efficacy of TsC [Tissue inhibitor of metalloproteinase-3 (TIMP3) + sulfated carboxymethylcellulose (sCMC)] and piperlongumine (PL) combination for the amelioration of OA in a goat ex vivo OA model. MAIN METHODS: The efficacy of the drug combination was evaluated using the goat ex vivo OA explant model and results were validated in clinically relevant human OA cartilage explants. The chondroprotective effects were evaluated in terms of reduced inflammation and cartilage matrix loss, reduction in chondrosenescence, and reduced oxidative stress. KEY FINDINGS: A combination of TsC and PL (TsC-PL) significantly reduced inflammation, cartilage matrix loss, chondrosenescence, and oxidative stress in the goat ex vivo OA model and showed chondroprotective effects. Further, similar chondroprotective effects were observed in human OA cartilage. Additionally, the coefficient of drug interaction analysis indicated that the combination of TsC and PL had a synergistic effect in reducing matrix degrading proteases and inflammation (goat ex vivo OA model) and Reactive oxygen species (ROS) production (human OA cartilage). SIGNIFICANCE: Combinatorial treatment with TsC and PL demonstrated potential disease-modifying effects for the treatment of osteoarthritis via inhibition of inflammation and senescence and supports the usage of treatment strategies targeting multiple pathological factors of OA simultaneously.

Converse modulation of Wnt/ß-catenin signaling during expansion and differentiation phases of Infrapatellar fat pad-derived MSCs for improved engineering of hyaline cartilage.

Mesenchymal stem cells (MSCs) are potential candidates in cell-based therapy for cartilage repair and regeneration. However, during chondrogenic differentiation, MSCs undergo undesirable hypertrophic maturation. This poses a risk of ossification in the neo-tissue formed that eventually impedes the clinical use of MSCs for cartilage repair. TGF-ß is a potent growth factor used for chondrogenic differentiation of MSCs, however, its role in hypertrophy remains ambiguous. In the present work, we decipher that TGF-ß activates Wnt/ß-catenin signaling through SMAD3 and increases the propensity of Infrapatellar fat pad derived MSCs (IFP-MSCs) towards hypertrophy. Notably, inhibiting TGF-ß induced Wnt/ß-catenin signaling suppresses hypertrophic progression and enhances chondrogenic ability of IFP-MSCs in plasma hydrogels. Additionally, we demonstrate that activating Wnt signaling during expansion phase, promotes proliferation and reduces senescence, while improving stemness of IFP-MSCs. Thus, conversely modulating Wnt signaling in vitro during expansion and differentiation phases generates hyaline-like cartilage with minimal hypertrophy. Importantly, pre-treatment of IFP-MSCs encapsulated in plasma hydrogel with Wnt modulators followed by subcutaneous implantation in nude mice resulted in formation of a cartilage tissue with negligible calcification. Overall, this study provides technological advancement on targeting Wnt/ß-catenin pathway in a 3D scaffold, while maintaining the standard chondro-induction protocol to overcome the challenges associated with the clinical use of MSCs to engineer hyaline cartilage.

Sulfated carboxymethylcellulose mediated enhancement of Timp3 efficacy synergistically attenuates osteoarthritis through inhibition of NFκB and JNK.

Osteoarthritis (OA) is a prevalent degenerative joint condition with no effective disease modifying treatments. In this study, we aimed to address multiple OA hallmarks using a combination of pro-chondrogenic sulfated carboxymethylcellulose (sCMC) and anti-catabolic tissue inhibitor of metalloproteases 3 (Timp3) in relevant disease systems. Firstly, we chemically sulfated carboxymethylcellulose to impart a negative charge and improve the stability of cationic Timp3. The modified sCMC exhibited a molecular weight of 10 kDa and a degree of sulfation of ∼10 %. We further demonstrated that sulfation of CMC confers pro-chondrogenic characteristics. Subsequently, we demonstrated that the combination of sCMC and Timp3 effectively reduced key OA hallmarks, such as matrix degradation, inflammation, and protease expression, in a goat ex vivo OA model compared to individual treatments. We further demonstrated that the anti-OA effect of sCMC and Timp3 is mediated through the suppression of NFκB and JNK activation. To validate the clinical potential and mechanism of action, we conducted experiments on human OA explants. The combination treatment synergistically reduced the expression of MMP13 and NFκB in human OA explants. Overall, sCMC-mediated enhancement of Timp3 efficacy synergistically reduced OA-like traits and demonstrates the potential for OA amelioration.

Facile synthesis of multi-faceted, biomimetic and cross-protective nanoparticle-based vaccines for drug-resistant Shigella: a flexible platform technology.

BACKGROUND: No commercial vaccines are available against drug-resistant Shigella due to serotype-specific/narrow-range of protection. Nanoparticle-based biomimetic vaccines involving stable, conserved, immunogenic proteins fabricated using facile chemistries can help formulate a translatable cross-protective Shigella vaccine. Such systems can also negate cold-chain transportation/storage thus overcoming challenges prevalent in various settings. METHODS: We explored facile development of biomimetic poly (lactide-co-glycolide)/PLGA 50:50 based nanovaccines (NVs), encapsulating conserved stabilized antigen(s)/immunostimulant of S. dysenteriae 1 origin surface-modified using simple chemistries. All encapsulants (IpaC/IpaB/LPS) and nanoparticles (NPs)-bare and modified (NV), were thoroughly characterized. Effect of IpaC on cellular uptake of NPs was assessed in-vitro. Immunogenicity of the NVs was assessed in-vivo in BALB/c mice by intranasal immunization. Cross-protective efficacy was assessed by intraperitoneally challenging the immunized groups with a high dose of heterologous S. flexneri 2a and observing for visible diarrhea, weight loss and survival. Passive-protective ability of the simplest NV was assessed in the 5-day old progeny of vaccinated mice. RESULTS: All the antigens and immunostimulant to be encapsulated were successfully purified and found to be stable both before and after encapsulation into NPs. The ~ 300 nm sized NPs with a zeta potential of ~ - 25 mV released ~ 60% antigen by 14th day suggesting an appropriate delivery kinetics. The NPs could be successfully surface-modified with IpaC and/or CpG DNA. In vitro experiments revealed that the presence of IpaC can significantly increase cellular uptake of NPs. All NVs were found to be cytocompatible and highly immunogenic. Antibodies in sera of NV-immunized mice could recognize heterologous Shigella. Immunized sera also showed high antibody and cytokine response. The immunized groups were protected from diarrhea and weight loss with ~ 70-80% survival upon heterologous Shigella challenge. The simplest NV showed ~ 88% survival in neonates. CONCLUSIONS: Facile formulation of biomimetic NVs can result in significant cross-protection. Further, passive protection in neonates suggest that parental immunization could protect infants, the most vulnerable group in context of Shigella infection. Non-invasive route of vaccination can also lead to greater patient compliance making it amenable for mass-immunization. Overall, our work contributes towards a yet to be reported platform technology for facile development of cross-protective Shigella vaccines.

Stable Recombinant Invasion Plasmid Antigen C (IpaC)-Based Single Dose Nanovaccine for Shigellosis.

Shigellosis, caused by the bacteria Shigella, is the leading cause of bacterial diarrhea and the second leading cause of diarrheal death among children under the age of five. Unfortunately, Shigella strains have acquired resistance to antibiotics, and a commercial vaccine is yet to be available. We have previously demonstrated that Shigella dysenteriae serotype 1 (Sd1)-based recombinant, stabilized, "invasion plasmid antigen C" (IpaC; 42 kDa) protein can induce robust immune responses in BALB/c mice against a challenge of a high dose of heterologous Shigella when immunized via three intranasal doses of IpaC without an adjuvant. In this work, in order to reduce the frequency of dosing and increase possible patient compliance, based on our previous screening, the minimum protective dose of stabilized IpaC (20 µg) was encapsulated in biodegradable polymeric poly(lactide-co-glycolide) nanoparticles (∼370 nm) and intranasally administered in BALB/c mice in a single dose. Interestingly, a single intranasal dose of the developed vaccine particles encapsulating only 20 µg of Sd1 IpaC led to a temporal increase in the antibody production with an improved cytokine response compared to free IpaC administered three times as described in our previous report. Upon intraperitoneal challenge with a high dose of heterologous Shigella flexneri 2a (common in circulation), the immunized animals were protected from diarrhea, lethargy, and weight loss with ∼67% survival, while all the control animals died by 36 h of the challenge. Overall, the developed nanovaccine could be explored as a potential noninvasive, cross-protective, single-dose, single-antigen Shigella vaccine amenable for scale-up and eventual mass immunization.

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint.

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Sulfated carboxymethylcellulose-based scaffold mediated delivery of Timp3 alleviates osteoarthritis.

Osteoarthritis (OA) is a debilitating progressive joint disease with high incidence and socioeconomic burden. However, no disease-modifying treatment is currently available for OA. Here, we report a sulfated carboxymethylcellulose-based scaffold mediated delivery of tissue inhibitor of metalloprotease 3 (Timp3) as a disease-modifying therapeutic strategy for OA. First, we chemically modified carboxymethylcellulose (CMC) to sulfated carboxymethylcellulose (sCMC) to impart native-like electrostatic interaction-based binding of cationic proteins. We then fabricated cartilage ECM mimicking sCMC-gelatin scaffolds which showed preferential binding and sustained delivery of Timp3. This scaffold-mediated delivery of Timp3 demonstrated a reduction in matrix degradation, protease expression and inflammatory markers in the goat ex vivo OA model leading to enhanced retention of cartilage ECM markers when compared to OA control. Further, similar results were obtained when sCMC-gelatin scaffolds were evaluated using human OA samples, supporting its clinical potential. Overall, the Timp3 loaded sCMC-gelatin scaffold shows potential as a treatment approach for OA.

A human osteoarthritis mimicking goat cartilage explant-based disease model for drug screening.

Although osteoarthritis (OA) is the most prevalent human joint disease with a large socioeconomic burden, it remains a neglected disease with no clinically approved disease modifying therapies. One of the key reasons for this is that the available disease models poorly recapitulate human OA-like traits, possibly because of the challenge of mimicking the disease in an ECM-rich cartilage tissue. In this study, we report the establishment and validation of a clinically relevant ex vivo OA model using IL1ß-treated goat articular cartilage explants. Treatment with IL1ß induced OA-like traits in goat cartilage explants and caused a shift in cartilage homeostasis towards enhanced catabolism, resulting in higher matrix degradation, overexpression of degradative and inflammatory mediators, and chondrocyte hypertrophy. We then validated the developed disease model for drug response using the drugs celecoxib, BMP7, and rapamycin, all of which demonstrated concentration-dependent disease amelioration in the model. Finally, we evaluated the translational relevance of the developed ex vivo OA model by comparing it with late-stage OA patient samples and observed a striking resemblance in terms of matrix degradation, expression of degradative enzymes, chondrocyte hypertrophy, and inflammation. Overall, the goat ex vivo OA model elicited a biological response to cytokine treatment that mirrors human OA-like traits and may reduce discordance between preclinical and clinical studies in OA drug development.

Bioinspired Injectable Hydrogels Dynamically Stiffen and Contract to Promote Mechanosensing-Mediated Chondrogenic Commitment of Stem Cells.

Developing stiff and resilient injectable hydrogels that can mechanically support load-bearing joints while enabling chondrogenic differentiation of stem cells is a major challenge in the field of cartilage tissue engineering. In the present work, a triple-network injectable hydrogel system was engineered using Bombyx mori silk fibroin, carboxymethyl cellulose (CMC), and gelatin. The developed hydrogel demonstrated a simultaneous increase in both stiffness and contraction over time, thereby imparting a four-dimensional (4D) evolving niche to the cells. While resilience was provided by CMC, the dynamic alterations in the hydrogel matrix were attributed to the formation of ß-sheets in silk. The engineered contraction facilitated condensation of cells that mimicked an important step during cartilage development. Subsequently, this led to downregulation of YAP signaling and enhanced chondrogenic commitment of stem cells. More importantly, the in vivo study showed that the ectopically regenerated cartilage was mature and closely resembled native articular cartilage. Overall, this strategy of engineering mechanotransduction that promotes chondrogenesis by contraction-mediated condensation is a promising and translatable approach for cartilage repair.

Carboxylated chitosan-mediated improved efficacy of mesoporous silica nanoparticle-based targeted drug delivery system for breast cancer therapy.

Nanoparticle-based targeting of overexpressed cell-surface receptors is a promising strategy that provides precise delivery of drugs to cancer cells. In the present study, we developed highly reproducible and monodispersed, chitosan-coated (pH-responsive), doxorubicin-loaded, aptamer-mesoporous silica nanoparticle (MSN) bioconjugates for actively targeting breast cancer cells harboring overexpression of EGF receptors (EGFR/HER2). The developed targeted MSNs demonstrated higher uptake and cytotoxicity of triple negative and HER2 positive breast cancer cells when compared to non-targeted MSNs. The chitosan coating imparted pH-responsiveness and endo/lysosomal escape ability to MSNs, which augmented cytosolic delivery of an anticancer drug. Partial carboxylation of chitosan coated on MSNs allowed for a greater release of drug in a shorter duration of time while retaining pH-responsiveness and endo/lysosomal escape ability. Overall, the coating of carboxylated-chitosan over MSNs enabled tunable drug release kinetics, conjugation of aptamers (targeting agents), and endo/lysosomal escape which together significantly enhanced the efficacy of the developed drug delivery system.

Development of a Self-Adjuvanting, Cross-Protective, Stable Intranasal Recombinant Vaccine for Shigellosis.

With the acquirement of antibiotic resistance, Shigella has resulted in multiple epidemics of shigellosis, an infectious diarrheal disease, causing thousands of deaths per year. Unfortunately, there are no licensed vaccines, primarily due to low or serotype-specific immunogenicity. Thus, conserved subunit vaccines utilizing recombinant invasion plasmid antigens (Ipa) have been explored as cross-protective vaccine candidates. However, achieving cross-protection against Shigella dysenteriae 1, which caused multiple pandemics/epidemics in the recent past, has been difficult. Therefore, a rational approach to improve cross-protection in the preparation for a possible pandemic should involve conserved proteins from S. dysenteriae 1 (Sd1). IpaC is one such conserved immunogenic protein that is less explored as an independent vaccine due to its instability/aggregation. Therefore, to improve cross-protection and potential immunogenicity and to be prepared for a future epidemic/pandemic, herein, we stabilized recombinant Sd1 IpaC, expressed without its chaperone, using a previously reported stabilizing detergent (LDAO) in a modified protocol and assessed its vaccine potential without an adjuvant. The protein assembled into heterogeneous complex spherical structures in the presence of LDAO and showed improved stability at storage temperatures of -80, -20, 4, 25, and 37 °C while providing enhanced yield and concentration. The protein could also be stably lyophilized and reconstituted, increasing the convenience of transportation and storage. Upon intranasal administration in BALB/c mice, the stabilized-IpaC-immunized groups generated significant antibody response and were not only protected against a high intraperitoneal dose of homologous S. dysenteriae 1 but also showed 100% survival against heterologous Shigella flexneri 2a without an adjuvant, while the control animals showed visible diarrhea (bloody-Sd1 challenge), lethargy, and weight loss with 0% survival. Overall, this work demonstrates that stabilized IpaC can be explored as a minimalist, self-adjuvanting, cross-protective, intranasal, single-antigen Shigella vaccine.

Simultaneous amelioration of diabetic ocular complications in lens and retinal tissues using a non-invasive drug delivery system.

Topically administered delivery systems for ophthalmic applications have been studied for the treatment of anterior or posterior eye diseases. However, simultaneous treatment of both anterior and posterior eye diseases has not been explored. In this study, we fabricated a topically administrable polymeric nanoparticle (NP)- based delivery system consisting of pluronic®F-68 shell and polycaprolactone core for the simultaneous treatment of both anterior and posterior eye diseases. These NPs were loaded with pyrrolidine dithiocarbamate (PDTC) or triamcinolone acetonide (TA) separately. The drug loading in NPs was optimized to initially achieve a moderate burst release of PDTC followed by slow and sustained release of both PDTC and TA. The resultant delivery system was studied for its in vivo efficacy in a diabetic retinopathy (DR) and cataract rat model. The results demonstrated that administration of PDTC NPs + TA NPs minimized oxidative stress in lens as evidenced by reduced levels of protein carbonyls and malondialdehyde, and, ameliorated DR complications in retina as evidenced by reduced expression of hypoxia inducible factor-1α along with a reduction in number of neovascular tufts and acellular capillaries. Therefore, delivery of PDTC and TA using PCL-PF68 NPs could be a useful approach for simultaneous treatment of diabetic cataract and DR.

A Synergistic Combination of Niclosamide and Doxorubicin as an Efficacious Therapy for All Clinical Subtypes of Breast Cancer.

Drug resistance is one of the major hurdles in the success of cancer chemotherapy. Notably, aberrantly expressed Wnt/ß-catenin signaling plays a major role in the initiation and maintenance of oncogenesis along with development of chemoresistance. Therefore, the combinatorial approach of targeting Wnt/ß-catenin pathway along with using a chemotherapeutic agent seems to be a promising strategy to improve cancer therapy. In the present study, we evaluated the combination of niclosamide (Nic), an FDA-approved antihelminthic drug repurposed as a Wnt signaling inhibitor, and doxorubicin (Dox), a conventional anticancer agent, in all clinical subtypes of breast cancer viz. triple negative breast cancer, HER2 positive breast cancer, and hormone receptor positive breast cancer. The results demonstrated that the combination induced apoptosis and caused synergistically enhanced death of all breast cancer cell types at multiple combinatorial concentrations using both the sequential and concurrent treatment regimens. Mechanistically, downregulation of Wnt/ß-catenin signaling and cell cycle arrest at G0/G1 phase by Nic and increase in reactive oxygen species by both Nic and Dox along with the inherent cytotoxicity of Dox mediated the synergism between the two drugs in both the treatment regimens. Overall, the combination of Nic and Dox holds promise to be developed as an efficient therapeutic option for breast cancer irrespective of its clinical subtype.

A non-invasive nanoparticle-based sustained dual-drug delivery system as an eyedrop for endophthalmitis.

Endophthalmitis is an infectious disease that affects the entire eye spreading to the internal retinal layers and the vitreous and causes severe sight-threatening conditions. Current treatment strategies rely on intraocular injections of antibiotics that are invasive, may lead to procedural complications and, ultimately, blindness. In this study, we developed a non-invasive strategy as an eyedrop containing nanoparticle-based dual-drug delivery system in which the hydrophobic poly-L-lactide core was loaded with azithromycin or triamcinolone acetonide, and the hydrophilic shell was made of chitosan. The developed nanoparticles were ~200-250 nm in size, spherical in shape, moderately hydrophilic, lysozyme tolerant, cytocompatible, and hemocompatible. Application of these chitosan-coated nanoparticles as eye drops to C57BL/6 mice showed higher bioavailability in choroid and retina when compared to the uncoated nanoparticles. The delivery system showed sustained release of drug for 300 h and exhibited antimicrobial effects against Gram-positive and Gram-negative bacteria and anti-inflammatory effects on activated microglial cells. Interestingly, the combination of the nanoparticles loaded with azithromycin and the nanoparticles loaded with triamcinolone acetonide acted synergistically as compared to either of the nanoparticles/drugs alone. Overall, the developed dual-drug delivery system is non-invasive, has antimicrobial and anti-inflammatory effects, and shows potential as an eye drop formulation against endophthalmitis.

Sulfated carboxymethylcellulose conjugated electrospun fibers as a growth factor presenting system for tissue engineering.

Inspired by the natural electrostatic interaction of cationic growth factors with anionic sulfated glycosaminoglycans in the extracellular matrix, we developed electrospun poly(hydroxybutyrate)/gelatin (PG) fibers conjugated with anionic sulfated carboxymethylcellulose (sCMC) to enable growth factor immobilization via electrostatic interaction for tissue engineering. The fibrous scaffold bound cationic molecules, was cytocompatible and exhibited a remarkable morphological and functional stability. Transforming growth factor-ß1 immobilized on the sCMC conjugated fibers was retained for at least 4 weeks with negligible release (3%). Immobilized fibroblast growth factor-2 and connective tissue growth factor were bioactive and induced proliferation and fibrogenic differentiation of infrapatellar fat pad derived mesenchymal stem cells respectively with efficiency similar to or better than free growth factors. Taken together, our studies demonstrate that sCMC conjugated PG fibers can immobilize and retain function of cationic growth factors and hence show potential for use in various tissue engineering applications.