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Antifibrotic and Pro-regenerative Effects of SMAD3 siRNA and Collagen I mRNA-Loaded Lipid Nanoparticles in Human Tenocytes
Abstract
Tendinopathy involves the inflammation and degeneration of the tendon due to repetitive strain injury. Current treatments primarily target inflammation resolution, yet they do not aim at tissue regeneration. In this study, a microfluidics approach is harnessed to develop a platform of lipid nanoparticles (LNPs) loaded simultaneously with SMAD3 siRNA and collagen I mRNA, aiming to explore its potential dual antifibrotic and regenerative effects in human tenocytes. The developed LNPs displayed size homogeneity and colloidal stability and exhibited high cytocompatibility in human tenocytes. Moreover, LNPs allowed for efficient uptake and transfection efficiency of the RNAs. In the in vitro efficacy studies, the gene expression and production of SMAD3 and collagen I were tested by real-time quantitative chain polymerase reaction and immuno- and intracellular staining, revealing collagen I production enhancement, SMAD3 inhibition, and modulation of other tendon repair factors by the LNPs. Overall, the potential of this platform of RNA-loaded LNPs to be used as a dual therapeutic approach to prevent fibrosis and promote tissue remodeling in late stages of tendon diseases was confirmed.
Tendon degeneration remains an intricate pathological process characterized by the coexistence of multiple dysregulated homeostasis processes, including the increase in collagen III production in comparison with collagen I. Mesenchymal stem...
The musculoskeletal system (MSKS) is composed of specialized connective tissues including bone, muscle, cartilage, tendon, ligament, and their subtypes. The primary function of the MSKS is to provide protection, structure, mobility, and mechanical properties to the body. In the process of fulfilling these functions, the MSKS is subject to wear and tear during aging and after injury and requires subsequent repair. MSKS diseases are a growing burden due to the increasing population age. The World Health Organization estimates that 1.71 billon people suffer from MSKS diseases worldwide. MSKS diseases usually involve various dysfunctions in bones, muscles, and joints, which often result in pain, disability, and a decrease in quality of life. The most common MSKS diseases are osteoporosis (loss of bone), osteoarthritis (loss of cartilage), and sarcopenia (loss of skeletal muscle). Because of the disease burden and the need for treatment, regenerative drug therapies for MSKS disorders are increasingly in demand. However, the difficulty of effective drug delivery in the MSKS has become a bottleneck for developing MSKS therapeutics. The abundance of extracellular matrix and its small pore size in the MSKS present a formidable barrier to drug delivery. Differences of vascularity among various MSKS tissues pose complications for drug delivery. Novel strategies are necessary to achieve successful drug delivery in different tissues composing the MSKS. Those considerations include the route of administration, mechanics of surrounding fluids, and biomolecular interactions, such as the size and charge of the particles and targeting motifs. This review focuses on recent advances in challenges to deliver drugs to each tissue of the MSKS, current strategies of drug delivery, and future ideas of how to overcome drug delivery challenges in the MSKS.
Macrophages play a key role in the development of many diseases, like tissue injury, cancer, and autoimmune diseases. So far, single‐drug loaded nanoparticles are developed to target macrophages. Nevertheless, macrophage dysregulation can induce multiple conditions, i.e., inflammation and fibrosis. Therefore, the simultaneous codelivery of a small molecule drug and a small interfering RNA (siRNA) for gene silencing may be beneficial to modulate macrophage dysfunction. Herein, hybrid lipid–polymer nanoparticles (LPNs) coloaded with both budesonide and enhanced green fluorescence protein siRNA (eGFP‐siRNA) as model anti‐inflammatory small molecule drug and siRNA, respectively, are developed by an optimized microfluidics method. Specifically, a poly(lactic‐co‐glycolic acid) core is coated by a lipid shell, and LPNs with size homogeneity and colloidal stability are obtained. Both payloads are loaded efficiently, and a controlled release is achieved. Additionally, LPNs are nontoxic in murine RAW 264.7 cells and human THP‐1 cells and are efficiently taken up by these cells. Finally, the transfection efficiency of dual‐loaded LPNs is high at low LPNs doses, thus proving the suitability of this nanosystem for gene silencing. Overall, the optimized LPNs are a suitable nanoplatform for the dual drug delivery to macrophages for the treatment of complex conditions requiring dual therapeutic approaches.
Tendinitis is a tendon disorder related to inflammation and pain, due to an injury or overuse of the tissue, which is hypocellular and hypovascular, leading to limited repair which occurs in a disorganized deposition of extracellular matrix that leads to scar formation and fibrosis, ultimately resulting in impaired tendon integrity. Current conventional treatments are limited and often ineffective, highlighting the need for new therapeutic strategies. In this work, acetalated-dextran nanoparticles (AcDEX NPs) loaded with curcumin and coated with tannic acid (TA) are developed to exploit the anti-inflammatory and anti-fibrotic properties of the two compounds. For this purpose, a microfluidic technique was used in order to obtain particles with a precise size distribution, aiming to decrease the batch-to-batch variability for possible future clinical translation. Coating with TA increased not only the stability of the nanosystem in different media but also enhanced the interaction and the cell-uptake in primary human tenocytes and KG-1 macrophages. The nanosystem exhibited good biocompatibility toward these cell types and a good release profile in an inflammatory environment. The efficacy was demonstrated by real-time quantitative polymerase chain reaction, in which the curcumin loaded in the particles showed good anti-inflammatory properties by decreasing the expression of NF-κb and TA-coated NPs showing anti-fibrotic effect, decreasing the gene expression of TGF-β. Overall, due to the loading of curcumin and TA in the AcDEX NPs, and their synergistic activity, this nanosystem has promising properties for future application in tendinitis.
Transforming growth factor-β is a key factor in regulating adhesion formation during tendon healing. We investigated the effectiveness of SMAD family member (Smad)7 and smad3 in the TGF-β/ Smad signaling during flexor tendon repair. Mouse flexor toe deep tendon rupture anastomosis models were made. On days 3, 7, 14, 21, and 28, the expressions of smad7 and smad3 in flexor tendon tissues were detected by RT-qPCR and western blot. Furthermore, postoperative intraperitoneal injections of smad7 agonists or smad3 antagonists were given. The degree of tendon healing was evaluated by adhesion testing and biomechanical experiments. HE staining was used to observe the pathological changes. Immunohistochemistry was used to evaluate the expressions of collagen III, smad3, and smad7. The mRNA levels of matrix metalloproteinase (mmp)2, mmp9, and scleraxis (SCX) in flexor tendon tissue were detected by RT-qPCR. Smad3 expression increased and smad7 expression decreased in flexor tendon tissue after injury. In addition, the smad7 agonist blocked smad3 phosphorylation. Smad7 agonist and smad3 antagonist both improved adhesion formation during flexor tendon healing, and decreased the expressions of collagen III, mmp9, and SCX, while increasing mmp2 expression. This study provides a possible theoretical basis for the smad7-smad3 signal cascade during flexor tendon adhesion healing.
Approved therapies for tendon diseases have not yet changed the clinical practice of symptomatic pain treatment and physiotherapy. This review article summarizes advances in the development of novel drugs, biologic products, and biomaterial therapies for tendon diseases with perspectives for translation of integrated therapies. Shifting from targeting symptom relief toward disease modification and prevention of disease progression may open new avenues for therapies. Deep evidence-based clinical, cellular, and molecular characterization of the underlying pathology of tendon diseases, as well as therapeutic delivery optimization and establishment of multidiscipline interorganizational collaboration platforms, may accelerate the discovery and translation of transformative therapies for tendon diseases.
Tendon injuries are at the frontier of innovative approaches to public health concerns and sectoral policy objectives. Indeed, these injuries remain difficult to manage due to tendon’s poor healing ability ascribable to a hypo-cellularity and low vascularity, leading to the formation of a fibrotic tissue affecting its functionality. Tissue engineering represents a promising solution for the regeneration of damaged tendons with the aim to stimulate tissue regeneration or to produce functional implantable biomaterials. However, any technological advancement must take into consideration the role of the immune system in tissue regeneration and the potential of biomaterial scaffolds to control the immune signaling, creating a pro-regenerative environment. In this context, immunoengineering has emerged as a new discipline, developing innovative strategies for tendon injuries. It aims at designing scaffolds, in combination with engineered bioactive molecules and/or stem cells, able to modulate the interaction between the transplanted biomaterial-scaffold and the host tissue allowing a pro-regenerative immune response, therefore hindering fibrosis occurrence at the injury site and guiding tendon regeneration. Thus, this review is aimed at giving an overview on the role exerted from different tissue engineering actors in leading immunoregeneration by crosstalking with stem and immune cells to generate new paradigms in designing regenerative medicine approaches for tendon injuries.
Statement of Retraction
Lei Han, Hong Liu, Huajun Fu, Yugen Hu, Weili Fang & Junsheng Liu - 2022, Exosome-delivered BMP-2 and polyaspartic acid promotes tendon bone healing in rotator cuff tear via Smad/RUNX2 signaling pathway, Bioengineered 13(1), doi: 10.1080/21655979.2021.2019871
Since publication, significant concerns have been raised about the integrity of the data and reported results in the article. When approached for an explanation, the authors did not provide their original data or any necessary supporting information. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. All authors listed in this publication have been informed.
We have been informed in our decision-making by our editorial policies and the COPE guidelines.
The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.
Recent years have witnessed incredible growth in RNA therapeutics, which has benefited significantly from decades of research on lipid nanoparticles, specifically its key component—the ionizable lipid. This comment discusses the major ionizable lipid types, and provides perspectives for future development. RNA therapeutics have benefited significantly from decades of research on lipid nanoparticles, specifically its key component—the ionizable lipid. This comment discusses the major ionizable lipid types, and provides perspectives for future development.
Abnormal tendons are rarely ever repaired to the natural structure and morphology of normal tendons. To better guide the repair and regeneration of injured tendons through a tissue engineering method, it is necessary to have insights into the internal morphology, organization, and composition of natural tendons. This review summarized recent researches on the structure and function of the extracellular matrix (ECM) components of tendons and highlight the application of multiple detection methodologies concerning the structure of ECMs. In addition, we look forward to the future of multi-dimensional biomaterial design methods and the potential of structural repair for tendon ECM components. In addition, focus is placed on the macro to micro detection methods for tendons, and current techniques for evaluating the extracellular matrix of tendons at the micro level are introduced in detail. Finally, emphasis is given to future extracellular matrix detection methods, as well as to how future efforts could concentrate on fabricating the biomimetic tendons.
Bone remodeling is a continuous process that maintains the homeostasis of the skeletal system, and it depends on the homeostasis between bone-forming osteoblasts and bone-absorbing osteoclasts. A large number of studies have confirmed that the Smad signaling pathway is essential for the regulation of osteoblastic and osteoclastic differentiation during skeletal development, bone formation and bone homeostasis, suggesting a close relationship between Smad signaling and bone remodeling. It is known that Smads proteins are pivotal intracellular effectors for the members of the transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMP), acting as transcription factors. Smad mediates the signal transduction in TGF-β and BMP signaling pathway that affects both osteoblast and osteoclast functions, and therefore plays a critical role in the regulation of bone remodeling. Increasing studies have demonstrated that a number of Smad signaling regulators have potential functions in bone remodeling. Therefore, targeting Smad dependent TGF-β and BMP signaling pathway might be a novel and promising therapeutic strategy against osteoporosis. This article aims to review recent advances in this field, summarizing the influence of Smad on osteoblast and osteoclast function, together with Smad signaling regulators in bone remodeling. This will facilitate the understanding of Smad signaling pathway in bone biology and shed new light on the modulation and potential treatment for osteoporosis.
COVID-19 vaccines have been developed with unprecedented speed which would not have been possible without decades of fundamental research on delivery nanotechnology. Lipid-based nanoparticles have played a pivotal role in the successes of COVID-19 vaccines and many other nanomedicines, such as Doxil ® and Onpattro ® , and have therefore been considered as the frontrunner in nanoscale drug delivery systems. In this review, we aim to highlight the progress in the development of these lipid nanoparticles for various applications, ranging from cancer nano-medicines to COVID-19 vaccines. The lipid-based nanoparticles discussed in this review are lipo-somes, niosomes, transfersomes, solid lipid nanoparticles, and nanostructured lipid carriers. We particularly focus on the innovations that have obtained regulatory approval or that are in clinical trials. We also discuss the physicochemical properties required for specific applications, highlight the differences in requirements for the delivery of different cargos, and introduce current challenges that need further development. This review serves as a useful guideline for designing new lipid nanoparticles for both preventative and therapeutic vaccines including immunotherapies.
Understanding the structure of messenger RNA lipid nanoparticles (mRNA-LNP) and specifically the microenvironment of the mRNA molecules within these entities is fundamental to advancing their biomedical potential. Here we show that a permeating cationic dye, thionine, can serve as a cryogenic electron microscopy (cryo-EM) contrasting agent by binding selectively to encapsulated mRNA without disturbing LNP morphology. Cryo-EM images identify the mRNA location revealing that mRNA may exist within solvent-filled cavities or may be substantially lipid-associated.
The clinical use of bioactive molecules in bone regeneration has been known to have side effects, which result from uncontrolled and supraphysiological doses. In this study, we demonstrated the synergistic effect of two bioactive molecules, bone morphogenic protein-2 (BMP-2) and alendronate (ALN), by releasing them in a sequential manner. Collagen-hydroxyapatite composite scaffolds functionalized using BMP-2 are loaded with biodegradable microspheres where ALN is encapsulated. The results indicate an initial release of BMP-2 for a few days, followed by the sequential release of ALN after two weeks. The composite scaffolds significantly increase osteogenic activity owing to the synergistic effect of BMP-2 and ALN. Enhanced bone regeneration was identified at eight weeks post-implantation in the rat 8-mm critical-sized defect. Our findings suggest that the sequential delivery of BMP-2 and ALN from the scaffolds results in a synergistic effect on bone regeneration, which is unprecedented. Therefore, such a system exhibits potential for the application of cell-free tissue engineering.
Tendinopathy describes a complex multifaceted pathology of the tendon, characterized by pain, decline in function and reduced exercise tolerance. The most common overuse tendinopathies involve the rotator cuff tendon, medial and lateral elbow epicondyles, patellar tendon, gluteal tendons and the Achilles tendon. The prominent histological and molecular features of tendinopathy include disorganization of collagen fibres, an increase in the microvasculature and sensory nerve innervation, dysregulated extracellular matrix homeostasis, increased immune cells and inflammatory mediators, and enhanced cellular apoptosis. Although diagnosis is mostly achieved based on clinical symptoms, in some cases, additional pain-provoking tests and imaging might be necessary. Management consists of different exercise and loading programmes, therapeutic modalities and surgical interventions; however, their effectiveness remains ambiguous. Future research should focus on elucidating the key functional pathways implicated in clinical disease and on improved rehabilitation protocols. Tendinopathy is a multifaceted pathology of the tendon, characterized by pain and reduced function. This Primer summarizes the epidemiology, pathophysiology, diagnosis and the latest insights in the management of this disorder. Finally, the authors discuss the outstanding issues that will help achieve better outcomes in patients with tendinopathy.
Background
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the resulting coronavirus disease 2019 (Covid-19) have afflicted tens of millions of people in a worldwide pandemic. Safe and effective vaccines are needed urgently.
Methods
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In an ongoing multinational, placebo-controlled, observer-blinded, pivotal efficacy trial, we randomly assigned persons 16 years of age or older in a 1:1 ratio to receive two doses, 21 days apart, of either placebo or the BNT162b2 vaccine candidate (30 μg per dose). BNT162b2 is a lipid nanoparticle–formulated, nucleoside-modified RNA vaccine that encodes a prefusion stabilized, membrane-anchored SARS-CoV-2 full-length spike protein. The primary end points were efficacy of the vaccine against laboratory-confirmed Covid-19 and safety.
Results
A total of 43,548 participants underwent randomization, of whom 43,448 received injections: 21,720 with BNT162b2 and 21,728 with placebo. There were 8 cases of Covid-19 with onset at least 7 days after the second dose among participants assigned to receive BNT162b2 and 162 cases among those assigned to placebo; BNT162b2 was 95% effective in preventing Covid-19 (95% credible interval, 90.3 to 97.6). Similar vaccine efficacy (generally 90 to 100%) was observed across subgroups defined by age, sex, race, ethnicity, baseline body-mass index, and the presence of coexisting conditions. Among 10 cases of severe Covid-19 with onset after the first dose, 9 occurred in placebo recipients and 1 in a BNT162b2 recipient. The safety profile of BNT162b2 was characterized by short-term, mild-to-moderate pain at the injection site, fatigue, and headache. The incidence of serious adverse events was low and was similar in the vaccine and placebo groups.
Conclusions
A two-dose regimen of BNT162b2 conferred 95% protection against Covid-19 in persons 16 years of age or older. Safety over a median of 2 months was similar to that of other viral vaccines. (Funded by BioNTech and Pfizer; ClinicalTrials.gov number, NCT04368728.)
In the recent of years, the use of lipid nanoparticles (LNPs) for RNA delivery has gained considerable attention, with a large number in the clinical pipeline as vaccine candidates or to treat a wide range of diseases. Microfluidics offers considerable advantages for their manufacture due to its scalability, reproducibility and fast preparation. Thus, in this study, we have evaluated operating and formulation parameters to be considered when developing LNPs. Among them, the flow rate ratio (FRR) and the total flow rate (TFR) have been shown to significantly influence the physicochemical characteristics of the produced particles. In particular, increasing the TFR or increasing the FRR decreased the particle size. The amino lipid choice (cationic—DOTAP and DDAB; ionisable—MC3), buffer choice (citrate buffer pH 6 or TRIS pH 7.4) and type of nucleic acid payload (PolyA, ssDNA or mRNA) have also been shown to have an impact on the characteristics of these LNPs. LNPs were shown to have a high (>90%) loading in all cases and were below 100 nm with a low polydispersity index (≤0.25). The results within this paper could be used as a guide for the development and scalable manufacture of LNP systems using microfluidics.
Percutaneous electrolysis is an emerging intervention proposed for the management of tendinopathies. Tendon pathology is characterized by a significant cell response to injury and gene expression. No study investigating changes in expression of those genes associated with collagen regeneration and remodeling of extracellular matrix has been conducted. The aim of this pilot study was to investigate gene expression changes after the application of percutaneous electrolysis on experimentally induced Achilles tendinopathy with collagenase injection in an animal model. Fifteen Sprague Dawley male rats were randomly divided into three different groups (no treatment vs. percutaneous electrolysis vs. needling). Achilles tendinopathy was experimentally induced with a single bolus of collagenase injection. Interventions consisted of 3 sessions (one per week) of percutaneous electrolysis or just needling. The rats were euthanized, and molecular expression of genes involved in tendon repair and remodeling, e.g., Cox2, Mmp2, Mmp9, Col1a1, Col3a1, Vegf and Scx, was examined at 28 days after injury. Histological tissue changes were determined with hematoxylin–eosin and safranin O analyses. The images of hematoxylin–eosin and Safranin O tissue images revealed that collagenase injection induced histological changes compatible with a tendinopathy. No further histological changes were observed after the application of percutaneous electrolysis or needling. A significant increase in molecular expression of Cox2, Mmp9 and Vegf genes was observed in Achilles tendons treated with percutaneous electrolysis to a greater extent than after just needling. The expression of Mmp2, Col1a1, Col3a1, or Scx genes also increased, but did not reach statistical significance. This animal study demonstrated that percutaneous electrolysis applied on an experimentally induced Achilles tendinopathy model could increase the expression of some genes associated with collagen regeneration and remodeling of extracellular matrix. The observed gene overexpression was higher with percutaneous electrolysis than with just needling.
Hereditary variant transthyretin amyloidosis (ATTRv) is a rare genetic defect that affects about 5000–10,000 people worldwide, causing amyloidosis secondary to misfolding of mutant transthyretin (TTR) protein fibrils. TTR mutations can cause protein deposits in many extracellular regions of organs, but those deposits in cardiac and axonal cells are the primary cause of this clinical syndrome. Treatment options are limited, but new drugs are being developed. Patisiran, a novel drug, is a liposomal siRNA against TTR that specifically targets this protein, reducing the accumulation of TTR in tissues, with subsequent improvement in both neuropathy and cardiac function. Patisiran is likely to serve as a prototype for the development of further intelligent drug solutions for use in targeted therapy. In this review we summarize the evidence currently available on the treatment of polyneuropathy in people with ATTRv with patisiran. We review the evidence on its efficacy, safety, and indications of use, citing novel and seminal papers on these subjects.
Tendon injuries are common with poor healing potential. The paucity of therapies for tendon injuries is due to our limited understanding of the cells and molecular pathways that drive tendon regeneration. Using a mouse model of neonatal tendon regeneration, we identified TGFβ signaling as a major molecular pathway that drives neonatal tendon regeneration. Through targeted gene deletion, small molecule inhibition, and lineage tracing, we elucidated TGFβ-dependent and TGFβ-independent mechanisms underlying tendon regeneration. Importantly, functional recovery depended on canonical TGFβ signaling and loss of function is due to impaired tenogenic cell recruitment from both Scleraxis-lineage and non-Scleraxis-lineage sources. We show that TGFβ signaling is directly required in neonatal tenocytes for recruitment and that TGFβ ligand is positively regulated in tendons. Collectively, these results show a functional role for canonical TGFβ signaling in tendon regeneration and offer new insights toward the divergent cellular activities that distinguish regenerative vs fibrotic healing.
Background:
Healing of tendons after injury involves the proliferation of tenocytes and the production of extracellular matrix; however, their capacity to heal is limited by poor cell density and limited growth factor activity. Flightless I (Flii) has previously been identified as an important regulator of cellular proliferation and migration, and the purpose of this study was to evaluate the effect of differential Flii gene expression on tenocyte function in vitro.
Methods:
The role of Flii on tenocyte proliferation, migration, and contraction was assessed using established assays. Tenocytes from Flii+/-, wild-type, and Flii overexpressing mice were obtained and the effect of differential Flii expression on migration, proliferation, contraction, and collagen synthesis determined in vitro. Statistical differences were determined using unpaired Student's t test and statistical outliers were identified using the Grubbs' test.
Results:
Flii overexpressing tenocytes showed significantly improved migration and proliferation as well as increased collagen I secretion. Explanted tendons from Flii overexpressing mice also showed significantly elevated tenocyte outgrowth compared to Flii+/- mice. In contrast to its role in dermal wound repair, Flii positively affects cellular processes in tendons.
Conclusions:
These findings suggest that Flii could be a novel target for modulating tenocyte activity and improving tendon repair. This could have significant clinical implications as novel therapeutic targets for improved healing of tendon injuries are urgently needed.
In this study, a rationally designed nanocomposite (BUDPDA@MAP) composed of polydopamine (PDA) nanoparticle and anti‐inflammatory drug budesonide (BUD) encapsulated in a pH‐responsive endosomolytic polymer (poly(butyl methacrylate‐co‐methacrylic acid) grafted acetalated dextran, denoted by MAP), is proposed. The uniform nanocomposite is prepared using a microfluidic device. At low endosomal pH (5.5), MAP destabilizes the endosomal membranes for the cytoplasmic delivery of PDA, and releases BUD simultaneously, resulting in a greater reactive oxygen species scavenging capability than both the free drug and PDA alone. The combined therapeutic efficacy from PDA and BUD also leads to a successful macrophage phenotype switch from pro‐inflammatory M1 to anti‐inflammatory M2.
The advent of nanomedicine has recently started to innovate the treatment of cardiovascular diseases, in particular myocardial infarction. Although current approaches are very promising, there is still urgent need for advanced targeting strategies. In this work, the exploitation of macrophages recruitment is proposed as novel and synergistic approach to improve the addressability of the infarcted myocardium achieved by current peptide-based heart targeting. For this purpose, an acetalated dextran-based nanosystem is designed and successfully functionalized with two different peptides, atrial natriuretic peptide (ANP) and linTT1, which are targeting, respectively, cardiac cells and macrophages associated to the atherosclerotic plaques. Biocompatibility of the nanocarrier is screened on both macrophages cell lines and primary macrophages, showing high safety, in particular after functionalization of the nanoparticles’ surface. Furthermore, the system shows higher association versus uptake ratio towards M2–like macrophages (approximately 2–fold and 6–fold increase in murine and human primary M2–like macrophages, respectively, compared to M1–like). Overall, the results demonstrate that the nanosystem has a potential ability to exploit the “hitchhike” effect on M2–like macrophages and potentially improve, in a dual targeting strategy, the ability of ANP peptide to target the infarcted heart.
Background:
Osteoarthritis and cartilage injury treatment is an unmet clinical need. Therefore, development of new approaches to treat these diseases is critically needed. Previous work in our laboratory has shown that murine muscle-derived stem cells (MDSCs) can efficiently repair articular cartilage in an osteochondral and osteoarthritis model. However, the cartilage repair capacity of human muscle-derived stem cells has not been studied which prompt this study.
Method:
In this study, we tested the in vitro chondrogenesis ability of six populations of human muscle-derived stem cells (hMDSCs), before and after lenti-BMP2/GFP transduction using pellet culture and evaluated chondrogenic differentiation of via histology and Raman spectroscopy. We further compared the in vivo articular cartilage repair of hMDSCs stimulated with BMP2 delivered through coacervate sustain release technology and lenti-viral gene therapy-mediated gene delivery in a monoiodoacetate (MIA)-induced osteoarthritis (OA) model. We used microCT and histology to evaluate the cartilage repair.
Results:
We observed that all hMDSCs were able to undergo chondrogenic differentiation in vitro. As expected, lenti-BMP2/GFP transduction further enhanced the chondrogenic differentiation capacities of hMDSCs, as confirmed by Alcian blue and Col2A1staining as well as Raman spectroscopy analysis. We observed through micro-CT scanning, Col2A1 staining, and histological analyses that delivery of BMP2 with coacervate could achieve a similar articular cartilage repair to that mediated by hMDSC-LBMP2/GFP. We also found that the addition of soluble fms-like tyrosine kinase-1 (sFLT-1) protein further improved the regenerative potential of hMDSCs/BMP2 delivered through the coacervate sustain release technology. Donor cells did not primarily contribute to the repaired articular cartilage since most of the repair cells are host derived as indicated by GFP staining.
Conclusions:
We conclude that the delivery of hMDSCs and BMP2 with the coacervate technology can achieve a similar cartilage repair relative to lenti-BMP2/GFP-mediated gene therapy. The use of coacervate technology to deliver BMP2/sFLT1 with hMDSCs for cartilage repair holds promise for possible clinical translation into an effective treatment modality for osteoarthritis and traumatic cartilage injury.
Self-amplifying RNA (saRNA) is a promising biotherapeutic tool that has been used as a vaccine against both infectious diseases and cancer. saRNA has been shown to induce protein expression for up to 60 days and elicit immune responses with lower dosing than messenger RNA (mRNA). Because saRNA is a large (~9500 nt), negatively charged molecule, it requires a delivery vehicle for efficient cellular uptake and degradation protection. Lipid nanoparticles (LNPs) have been widely used for RNA formulations, where the prevailing paradigm is to encapsulate RNA within the particle, including the first FDA-approved small-interfering siRNA therapy. Here, we compared LNP formulations with cationic and ionizable lipids with saRNA either on the interior or exterior of the particle. We show that LNPs formulated with cationic lipids protect saRNA from RNAse degradation, even when it is adsorbed to the surface. Furthermore, cationic LNPs deliver saRNA equivalently to particles formulated with saRNA encapsulated in an ionizable lipid particle, both in vitro and in vivo. Finally, we show that cationic and ionizable LNP formulations induce equivalent antibodies against HIV-1 Env gp140 as a model antigen. These studies establish formulating saRNA on the surface of cationic LNPs as an alternative to the paradigm of encapsulating RNA.
The current pilot study investigates whether oral supplementation of specific collagen peptides improves symptoms and tendon vascularisation in patients with chronic mid-portion Achilles tendinopathy in combination with structured exercise. Participants were given a placebo or specific collagen peptides (TENDOFORTE®) in combination with a bi-daily calf-strengthening program for 6 months. Group AB received specific collagen peptides for the first 3 months before crossing over to placebo. Group BA received placebo first before crossing over to specific collagen peptides. At baseline (T1), 3 (T2) and 6 (T3) months, Victorian Institute of Sports Assessment–Achilles (VISA-A) questionnaires and microvascularity measurements through contrast-enhanced ultrasound were obtained in 20 patients. Linear mixed modeling statistics showed that after 3 months, VISA-A increased significantly for group AB with 12.6 (9.7; 15.5), while in group BA VISA-A increased only by 5.3 (2.3; 8.3) points. After crossing over group AB and BA showed subsequently a significant increase in VISA-A of, respectively, 5.9 (2.8; 9.0) and 17.7 (14.6; 20.7). No adverse advents were reported. Microvascularity decreased in both groups to a similar extent and was moderately associated with VISA-A (Rc2:0.68). We conclude that oral supplementation of specific collagen peptides may accelerate the clinical benefits of a well-structured calf-strengthening and return-to-running program in Achilles tendinopathy patients.
Background
The genes of SPARC, CCR2, and SMAD3 are implicated in orchestrating inflammatory response that leads to fibrosis in scleroderma and other fibrotic disorders. The aim of the studies is to evaluate synergistic anti-fibrotic potency of the siRNAs of these genes.
Methods
The efficacy of the siRNA-combination was evaluated in bleomycin-induced mouse fibrosis. The pathological changes of skin and lungs of the mice were assessed by hematoxylin and eosin and Masson's trichrome stains. The expression of inflammation and fibrosis associated genes and proteins in the tissues were assessed by real-time RT-PCR, RNA sequencing, Western blots and ELISA. Non-crosslinked fibrillar collagen was measured by the Sircol colorimetric assay.
Findings
The applications of the combined siRNAs in bleomycin-induced mice achieved favorable anti-inflammatory and anti-fibrotic effects. Activation of fibroblasts was suppressed in parallel with inhibition of inflammation evidenced by reduced inflammatory cells and proinflammatory cytokines in the BALF and/or the tissues by the treatment. Aberrant expression of the genes normally expressed in fibroblasts, monocytes/ macrophage, endothelial and epithelial cells were significantly restrained after the treatment. In addition, transcriptome profiles indicated that some bleomycin-induced alterations of multiple biological pathways were recovered to varying degrees by the treatment.
Interpretation
The application of the combined siRNAs of SPARC, CCR2, and SMAD3 genes ameliorated inflammation and fibrosis in bleomycin-induced mice. It systemically reinstated multiple biopathways, probably through controlling on different cell types including fibroblasts, monocytes/macrophages, endothelial cells and others. The multi-target-combined therapeutic approach examined herein may represent a novel and effective therapy for fibrosis.
Background
The Global Burden of Diseases, Injuries, and Risk Factors Study 2017 (GBD 2017) includes a comprehensive assessment of incidence, prevalence, and years lived with disability (YLDs) for 354 causes in 195 countries and territories from 1990 to 2017. Previous GBD studies have shown how the decline of mortality rates from 1990 to 2016 has led to an increase in life expectancy, an ageing global population, and an expansion of the non-fatal burden of disease and injury. These studies have also shown how a substantial portion of the world's population experiences non-fatal health loss with considerable heterogeneity among different causes, locations, ages, and sexes. Ongoing objectives of the GBD study include increasing the level of estimation detail, improving analytical strategies, and increasing the amount of high-quality data.
Methods
We estimated incidence and prevalence for 354 diseases and injuries and 3484 sequelae. We used an updated and extensive body of literature studies, survey data, surveillance data, inpatient admission records, outpatient visit records, and health insurance claims, and additionally used results from cause of death models to inform estimates using a total of 68 781 data sources. Newly available clinical data from India, Iran, Japan, Jordan, Nepal, China, Brazil, Norway, and Italy were incorporated, as well as updated claims data from the USA and new claims data from Taiwan (province of China) and Singapore. We used DisMod-MR 2.1, a Bayesian meta-regression tool, as the main method of estimation, ensuring consistency between rates of incidence, prevalence, remission, and cause of death for each condition. YLDs were estimated as the product of a prevalence estimate and a disability weight for health states of each mutually exclusive sequela, adjusted for comorbidity. We updated the Socio-demographic Index (SDI), a summary development indicator of income per capita, years of schooling, and total fertility rate. Additionally, we calculated differences between male and female YLDs to identify divergent trends across sexes. GBD 2017 complies with the Guidelines for Accurate and Transparent Health Estimates Reporting.
Findings
Globally, for females, the causes with the greatest age-standardised prevalence were oral disorders, headache disorders, and haemoglobinopathies and haemolytic anaemias in both 1990 and 2017. For males, the causes with the greatest age-standardised prevalence were oral disorders, headache disorders, and tuberculosis including latent tuberculosis infection in both 1990 and 2017. In terms of YLDs, low back pain, headache disorders, and dietary iron deficiency were the leading Level 3 causes of YLD counts in 1990, whereas low back pain, headache disorders, and depressive disorders were the leading causes in 2017 for both sexes combined. All-cause age-standardised YLD rates decreased by 3·9% (95% uncertainty interval [UI] 3·1–4·6) from 1990 to 2017; however, the all-age YLD rate increased by 7·2% (6·0–8·4) while the total sum of global YLDs increased from 562 million (421–723) to 853 million (642–1100). The increases for males and females were similar, with increases in all-age YLD rates of 7·9% (6·6–9·2) for males and 6·5% (5·4–7·7) for females. We found significant differences between males and females in terms of age-standardised prevalence estimates for multiple causes. The causes with the greatest relative differences between sexes in 2017 included substance use disorders (3018 cases [95% UI 2782–3252] per 100 000 in males vs s1400 [1279–1524] per 100 000 in females), transport injuries (3322 [3082–3583] vs 2336 [2154–2535]), and self-harm and interpersonal violence (3265 [2943–3630] vs 5643 [5057–6302]).
Interpretation
Global all-cause age-standardised YLD rates have improved only slightly over a period spanning nearly three decades. However, the magnitude of the non-fatal disease burden has expanded globally, with increasing numbers of people who have a wide spectrum of conditions. A subset of conditions has remained globally pervasive since 1990, whereas other conditions have displayed more dynamic trends, with different ages, sexes, and geographies across the globe experiencing varying burdens and trends of health loss. This study emphasises how global improvements in premature mortality for select conditions have led to older populations with complex and potentially expensive diseases, yet also highlights global achievements in certain domains of disease and injury.
Background/aims
The contribution of inflammation to tendinopathy has been debated in the scientific literature. Several factors may contribute to this lack of clarity, including inconsistent definitions of inflammation. We hypothesised that the adoption and/or rejection of a causal link between inflammation and tendinopathy varied as a function of the ‘inflammatory component’ (eg, immune cell and molecular mediators included in published reviews).
Methods
Twenty data items were collected from each review to determine conclusions about the role of inflammation in tendinopathy, specific definitions of the ‘inflammatory component,’ quality of the review and other potential correlates. Associations between correlates and a review’s conclusion about the role of inflammation in tendinopathy were tested using binomial logistic regression. The database searches retrieved 2261 unique publications: 137 fulfilled inclusion criteria after full text screenings.
Results
There has been little support for an inflammatory component to tendinopathy until recently (2012–2015). Prior to 2012, the majority of published reviews did not discuss monocytes, macrophages or lymphocytes in tendinopathy; rather they focused on the lack of neutrophils, often referred to as ‘the inflammatory infiltrate’, or immune cells were not discussed. Reviews including monocytes and lymphocytes in their discussions were 5.23 times more likely to conclude inflammation was important than reviews that did not, p<0.001.
Conclusions
Data collected show growing support for an inflammatory component to tendinopathy, particularly among high-quality reviews and those that used more robust definitions of inflammation. This finding may have implications for explaining dissonance in the literature regarding a causal role for inflammation in the pathogenesis of tendinopathy.
Tendinopathy is a common disease of the musculoskeletal system, particularly in athletes and sports amateurs. In this review, we will present evidence for the critical role of inflammatory mediators and immunocytes in the pathogenesis of tendinopathy and the efficacy of current antiinflammatory therapy and regenerative medicine in the clinic. We hereby propose a hypothesis that in addition to pulling force there may be compressive forces being exerted on the tendon during physical activities, which may initiate the onset of tendinopathy.
The translational potential of this article: Understanding the mechanisms of inflammation and existing antiinflammatory and regenerative therapies is key to the development of therapeutic strategies in tendinopathy.
We performed literature searches on MEDLINE from the inception of this review to February 2018. No language restrictions were imposed. The search terms were as follows: ("Tendinopathy"[Mesh] OR "Tendon Injuries"[Mesh] OR "Tendinitis"[Mesh] OR "Tendon"[Mesh]) AND (Inflammation OR "Inflammatory mediator*" OR Immunocyte*) OR ("anti inflammatory*" OR "regenerative medicine"). Inclusion criteria included articles that were original and reliable, with the main contents being highly relevant to our review. Exclusion criteria included articles that were not available online or have not been published. We scanned the abstract of these articles first. This was then followed by a careful screening of the articles which might be suitable for our review. Finally, 84 articles were selected as references. This review article is written in the narrative form.
Both acute and chronic tendinopathy result in high morbidity, requiring management that is often lengthy and expensive. However, limited and conflicting scientific evidence surrounding current management options has presented a challenge when trying to identify the best treatment for tendinopathy. As a result of shortcomings of current treatments, response to available therapies is often poor, resulting in frustration in both patients and physicians. Due to a lack of understanding of basic tendon-cell biology, further scientific investigation is needed in the field for the development of biological solutions. Optimization of new delivery systems and therapies that spatially and temporally mimic normal tendon physiology hold promise for clinical application. This review focuses on the clinical importance of tendinopathy, the structure of healthy tendons, tendon injury, and healing, and a discussion of current approaches for treatment that highlight the need for the development of new nonsurgical interventions.
Biohybrid nanosystems are at the center of personalized medicine, affording prolonged
circulation time and targeting to the disease site, and serving as antigenic sources of vaccines. The optimization and functionality parameters of these nanosystems vary depending on the properties of the core particles. In this work, the effects of the core particles’ surface charge and hydrophobicity are evaluated on the nanosystem coating with vesicles derived from plasma membrane. The measured parameters are the dimensions, surface charge, shape, and stability of the biohybrid nanosystems, both in buffer and in biologically relevant media (plasma and simulated synovial fluid). Moreover, the cytocompatibility properties of the developed nanosystems are evaluated in different cell lines mimicking the target cell populations and other districts of the body involved in the distribution and elimination of the nanoparticles. Finally, the immunological profile of the particles is investigated, highlighting the absence of immune activation promoted by the nanoplatforms.
Background
Tendon disease is characterized by the development of fibrosis. Transforming growth factor beta (TGF-β), bone morphogenic proteins (BMPs) and connective tissue growth factor (CTGF) are key mediators in the pathogenesis of fibrotic disorders. The aim of this systematic review was to investigate the evidence for the expression of TGF-β, BMPs and CTGF along tendon disease progression and the response of tendon cells to these growth factors accordingly. Method
We conducted a systematic screen of the scientific literature using the Medline database. The search terms used were “tendon AND TGF-β,” “tendon AND BMP” or “tendon AND CTGF.” Studies of human samples, animal tendon injury and overuse models were included. ResultsThirty-three studies were included. In eight studies the expression of TGF-β, BMPs or CTGF was dysregulated in chronic tendinopathy and tendon tear patient tissues in comparison with healthy control tissues. The expression of TGF-β, BMPs and CTGF was increased and showed temporal changes in expression in tendon tissues from animal injury or overuse models compared with the healthy control (23 studies), but the pattern of upregulation was inconsistent between growth factors and also the type of animal model. No study investigated the differences in the effect of TGF-β, BMPs or CTGF treatment between patient-derived cells from healthy and diseased tendon tissues. Tendon cells derived from animal models of tendon injury showed increased expression of extracellular matrix protein genes and increased cell signaling response to TGF-β and BMP treatments compared with the control cells (two studies). Conclusion
The expression of TGF-β, BMPs and CTGF in tendon tissues is altered temporally during healing in animal models of tendon injury or overuse, but the transition during the development of human tendon disease is currently unknown. Findings from this systematic review suggest a potential and compelling role for TGF-β, BMPs and CTGF in tendon disease; however, there is a paucity of studies analyzing their expression and stimulated cellular response in well-phenotyped human samples. Future work should investigate the dynamic expression of these fibrotic growth factors and their interaction with tendon cells using patient samples at different stages of human tendon disease.
Differential mRNA expression studies implicitly assume that changes in mRNA expression have biological meaning, most likely mediated by corresponding changes in protein levels. Yet studies into mRNA-protein correspondence have shown notoriously poor correlation between mRNA and protein expression levels, creating concern for inferences from only mRNA expression data. However, none of these studies have examined in particular differentially expressed mRNA. Here, we examined this question in an ovarian cancer xenograft model. We measured protein and mRNA expression for twenty-nine genes in four drug-treatment conditions and in untreated controls. We identified mRNAs differentially expressed between drug-treated xenografts and controls, then analysed mRNA-protein expression correlation across a five-point time-course within each of the four experimental conditions. We evaluated correlations between mRNAs and their protein products for mRNAs differentially expressed within an experimental condition compared to those that are not. We found that differentially expressed mRNAs correlate significantly better with their protein product than non-differentially expressed mRNAs. This result increases confidence for the use of differential mRNA expression for biological discovery in this system, as well as providing optimism for the usefulness of inferences from mRNA expression in general.
In vitro transcribed (IVT) mRNA has recently come into focus as a potential new drug class to deliver genetic information. Such synthetic mRNA can be engineered to transiently express proteins by structurally resembling natural mRNA. Advances in addressing the inherent challenges of this drug class, particularly related to controlling the translational efficacy and immunogenicity of the IVTmRNA, provide the basis for a broad range of potential applications. mRNA-based cancer immunotherapies and infectious disease vaccines have entered clinical development. Meanwhile, emerging novel approaches include in vivo delivery of IVT mRNA to replace or supplement proteins, IVT mRNA-based generation of pluripotent stem cells and genome engineering using IVT mRNA-encoded designer nucleases. This Review provides a comprehensive overview of the current state of mRNA-based drug technologies and their applications, and discusses the key challenges and opportunities in developing these into a new class of drugs.
The surfaces of nanoparticles become covered by biomolecules in biological fluids. This protein ‘corona’ modifies materials’ characteristics and biological activity. The composition of the protein corona is dynamic, abundant biomolecules that bind first are subsequently replaced by less abundant but more tightly bound ones. Here, we explore the formation of the silver nanoparticle protein corona on exposure to cell culture media containing 10% fetal bovine serum supplemented Dulbecco's Modified Eagle's medium. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry analysis were used to monitor how different parameters such as incubation time, heating duration, cell culture medium, incubation temperature, and the number of washes affect the nanoparticle–protein corona complex. silver nanoparticles with and without bound proteins were characterized by electron microscopy, dynamic light scattering, and ultraviolet-visible-near-IR spectroscopy. The tetrazolium-based MTT assay was used to determine viability of A549 human lung adenocarcinoma cells treated with silver nanoparticles. Characterization of the nanoparticles before and after protein binding provided insights into their changing morphology on corona formation. Our results confirmed that the physiological environment directly affects protein corona formation on nanoparticle surfaces. In particular, incubation condition-dependent differences in the amount of bound proteins were observed. This work highlights the importance of environmental drivers of protein adsorption, which should be considered when predicting and/or controlling protein targets of silver nanoparticles.
Messenger RNA (mRNA) has emerged as a new category of therapeutic agent to prevent and treat various diseases. To function in vivo, mRNA requires safe, effective and stable delivery systems that protect the nucleic acid from degradation and that allow cellular uptake and mRNA release. Lipid nanoparticles have successfully entered the clinic for the delivery of mRNA; in particular, lipid nanoparticle–mRNA vaccines are now in clinical use against coronavirus disease 2019 (COVID-19), which marks a milestone for mRNA therapeutics. In this Review, we discuss the design of lipid nanoparticles for mRNA delivery and examine physiological barriers and possible administration routes for lipid nanoparticle–mRNA systems. We then consider key points for the clinical translation of lipid nanoparticle–mRNA formulations, including good manufacturing practice, stability, storage and safety, and highlight preclinical and clinical studies of lipid nanoparticle–mRNA therapeutics for infectious diseases, cancer and genetic disorders. Finally, we give an outlook to future possibilities and remaining challenges for this promising technology.
Owing to specific and compelling gene silencing, RNA interference (RNAi) is expected to become an essential approach in treating a variety of infectious, hemato-oncological, cardiovascular, and neurodegenerative conditions. The mechanism of action of small interfering RNA (siRNA) is based on post-transcriptional gene silencing. siRNA molecules are usually specific and efficient in the knockdown of disease-related genes. However, they are characterized by low cellular uptake and are susceptible to nuclease-mediated degradation. Therefore, siRNAs require a carrier for their protection and efficient delivery into target cells. The current review highlights the siRNA-based mechanism of action, challanges, and recent advances in clinical applications.
Delivering nucleic acid-based therapeutics to cells is an attractive approach to target the genetic cause of various diseases. In contrast to conventional small molecule drugs that target gene products (i.e., proteins), genetic drugs induce therapeutic effects by modulating gene expression. Gene silencing, the process whereby protein production is prevented by neutralizing its mRNA template, is a potent strategy to induce therapeutic effects in a highly precise manner. Importantly, gene silencing has broad potential as theoretically any disease-causing gene can be targeted. It was demonstrated two decades ago that introducing synthetic small interfering RNAs (siRNAs) into the cytoplasm results in specific degradation of complementary mRNA via a process called RNA interference (RNAi). Since then, significant efforts and investments have been made to exploit RNAi therapeutically and advance siRNA drugs to the clinic. Utilizing (unmodified) siRNA as a therapeutic, however, is challenging due to its limited bioavailability following systemic administration. Nuclease activity and renal filtration result in siRNA's rapid clearance from the circulation and its administration induces (innate) immune responses. Furthermore, siRNA's unfavorable physicochemical characteristics largely prevent its diffusion across cellular membranes, impeding its ability to reach the cytoplasm where it can engage the RNAi machinery. The clinical translation of siRNA therapeutics has therefore been dependent on chemical modifications and developing sophisticated delivery platforms to improve their stability, limit immune activation, facilitate internalization, and increase target affinity. These developments have resulted in last year's approval of the first siRNA therapeutic, called Onpattro (patisiran), for treatment of hereditary amyloidogenic transthyretin (TTR) amyloidosis. This disease is characterized by a mutation in the gene encoding TTR, a serum protein that transports retinol in circulation following secretion by the liver. The mutation leads to production of misfolded proteins that deposit as amyloid fibrils in multiple organs, resulting in progressive neurodegeneration. Patisiran's therapeutic effect relies on siRNA-mediated TTR gene silencing, preventing mutant protein production and halting or even reversing disease progression. For efficient therapeutic siRNA delivery to hepatocytes, patisiran is critically dependent on lipid nanoparticle (LNP) technology. In this Account, we provide an overview of key advances that have been crucial for developing LNP delivery technology, and we explain how these developments have contributed to the clinical translation of siRNA therapeutics for parenteral administration. We discuss optimization of the LNP formulation, particularly focusing on the rational design of ionizable cationic lipids and poly(ethylene glycol) lipids. These components have proven to be instrumental for highly efficient siRNA encapsulation, favorable LNP pharmacokinetic parameters, and hepatocyte internalization. Additionally, we pay attention to the development of rapid mixing-based methods that provide robust and scalable LNP production procedures. Finally, we highlight patisiran's clinical translation and LNP delivery technology's potential to enable the development of genetic drugs beyond the current state-of-the-art, such as mRNA and gene editing therapeutics.
Tendon injuries are common and can dramatically impair patient mobility and productivity, resulting in a significant socioeconomic burden and reduced quality of life. Because the tendon healing process results in the formation of a fibrotic scar, injured tendons never regain the mechanical strength of the uninjured tendon, leading to frequent reinjury. Many tendons are also prone to the development of peritendinous adhesions and excess scar formation, which further reduce tendon function and lead to chronic complications. Despite this, there are currently no treatments that adequately improve the tendon healing process due in part to a lack of information regarding the contributions of various cell types to tendon healing and how their activity may be modulated for therapeutic value. In this review, we summarize recent efforts to identify and characterize the distinct cell populations involved at each stage of tendon healing. In addition, we examine the mechanisms through which different cell populations contribute to the fibrotic response to tendon injury, and how these responses can be affected by systemic factors and comorbidities. We then discuss gaps in our current understanding of tendon fibrosis and highlight how new technologies and research areas are shedding light on this clinically important and intractable challenge. A better understanding of the complex cellular environment during tendon healing is crucial to the development of new therapies to prevent fibrosis and promote tissue regeneration.
Introduction: Nanoparticles are anticipated to overcome persistent challenges in efficient drug delivery, but the limitations associated with conventional methods of preparation are resulting in slow translation from research to clinical applications. Due to their enormous potential, microfluidic technologies have emerged as an advanced approach for the development of drug delivery systems with well-defined physicochemical characteristics and in a reproducible manner.
Areas covered: This review provides an overview of microfluidic devices and materials used for their manufacturing, together with the flow patterns and regimes commonly used for nanoparticle preparation. Additionally, the different geometries used in droplet microfluidics are reviewed, with particular attention to the co-flow geometry used for the production of nanoparticles. Finally, this review summarizes the main and most recent nanoparticulate systems prepared using microfluidics, including drug nanosuspensions, polymeric, lipid, structured, and theranostic nanoparticles.
Expert opinion: The production of nanoparticles at industrial scale is still a challenge, but the microfluidic technologies bring exciting opportunities to develop drug delivery systems that can be engineered in an easy, cost-effective and reproducible manner. As a highly interdisciplinary research field, more efforts and general acceptance are needed to allow for the translation of nanoparticulate drug delivery systems from academic research to the clinical practice.
In recent years, biomaterials have gained importance as vehicles and adjuvants in the
formulation of novel cancer vaccines. Among the different biomaterials proposed
porous silicon (PSi) provides a versatile biocompatible platform for various biomedical
applications. The aim of this work was to develop a multistage nanovaccine
constructed from biomaterials and a biological cancer cell membrane (CCM), and to
assess its immunostimulative properties. Glass-capillary microfluidic nanoprecipitation
technique was used to produce the initial two layers of a nanovaccine. TOPSi
nanoparticles were encapsulated into acetalated dextran (AcDEX) or sperminemodified
AcDEX (SpAcDEX) and further co-extruded together with vesicles derived
from CCM to obtain the final core-shell system (TOPSi@AcDEX@CCM), in order to
combine the antigenic composition of tumor lysate with the adjuvant properties of the
PSi nanoparticles chosen. TOPSi@SpAcDEX particles were further functionalized with
a model antigen, Trp2, to provide the final system, TOPSi@SpAcDEX-Trp2, for
targeting. The nanovaccines presented high monodispersity and cytocompatibility,
induced expression of co-stimulatory signals in both immortal cell lines and in
peripheral blood monocytes, and enhanced the secretion of IFN-γ. Overall, the
developed nanovaccines showed promising adjuvant properties and the possibility of
encapsulating the nanosystems with materials derived from the patient's tumor cells
opens new prospectives for personalized cancer therapy.
Tendon adhesion is a common problem in the healing of injured tendons. The molecular mechanisms of the TGF-β/Smad signaling pathway have been determined, and the role of TGF-β has been well characterized in wound healing. However, the intracellular mechanism or downstream signals by which TGF-β3 modulates its effects on tendon healing have not been well elucidated. The aim of this study was to determine the effect of TGF‑β3 on the TGF-β/Smad signaling pathway in tenocytes. Quantitative polymerase chain reaction and western blot analysis were used to analyze the effect of TGF‑β3 on the regulation of the expression of Smad proteins in tenocytes. The results demonstrated that TGF‑β3 has no significant effect on the proliferation of tendon cells. The addition of TGF‑β3 to tenocytes can significantly downregulate the expression of Smad3 and upregulate the expression of Smad7 at the gene and protein levels. The results demonstrate that TGF‑β3 may regulate Smad3 and Smad7 proteins through the TGF-β/Smad signaling pathway to minimize extrinsic scarring. Thus, it may provide a novel approach to decrease tendon adhesion and promote tendon healing.
Purpose:
Preclinical studies have reported that bone morphogenetic protein (BMP)-2 promotes bone-tendon healing following anterior cruciate ligament reconstruction. We examined the region-specific effects of BMP-2 on osteoblast and fibroblast differentiation in a highly standardized murine in vitro co-culture model of bone-tendon integration.
Materials and methods:
We used quantitative PCR to measure the dose-and time-dependent influence of BMP-2 on the expression of alkaline phosphatase, osteocalcin, collagen type 1 (alpha 1 chain), runt-related transcription factor 2, osteopontin, collagen type 1 (alpha 2 chain), collagen type 5 (alpha 1 chain), decorin, fibromodulin, mohawk homeobox, bone morphogenetic protein receptor, type 1A, bone morphogenetic protein receptor, type 2 and noggin in the osteoblast, interface and fibroblast regions of a co-culture model of the murine preosteoblast cell line MC3T3-E1 and the fibroblast cell line 3T6.
Results:
Stimulation with BMP-2 caused a significant upregulation of alkaline phosphatase (p <0.001), osteocalcin (p <0.001), collagens (p <0.001), runt-related transcription factor 2 (p <0.05) and osteopontin (p <0.001) expression in the osteoblast region. In the interface region, BMP-2 exposure led to dose- and time-dependent upregulation of alkaline phosphatase (p <0.001), osteocalcin (p <0.001), osteopontin (p <0.001), runt-related transcription factor 2 (p <0.001) and markers of extracellular matrix production (p <0.001). Both, BMP receptors showed a significant BMP-2 dependent upregulation at the interface region, and Noggin was downregulated at the osteoblast and interface region following BMP-2 exposure.
Conclusions:
Exposure to BMP-2 upregulated the expression of genes associated with bone-tendon integration in vitro, suggesting the stimulation of transdifferentiation processes at the interface and fibroblast regions as well as the induction of positive feedback mechanisms. Further studies will be needed to establish BMP-2 dose and treatment algorithms following tendon reinsertion and reconstruction.
Flexor tendons (FT) in the hand provide near frictionless gliding to facilitate hand function. Upon injury and surgical repair, satisfactory healing is hampered by fibrous adhesions between the tendon and synovial sheath. In the present study we used antisense oligonucleotides (ASOs), specifically targeted to components of Tgf-β signaling, including Tgf-β1, Smad3 and Ctgf, to test the hypothesis that local delivery of ASOs and suppression of Tgf-β1 signaling would enhance murine FT healing by suppressing adhesion formation while maintaining strength. ASOs were injected in to the FT repair site at 2, 6 and 12 days post-surgery. ASO treatment suppressed target gene expression through 21 days. Treatment with Tgf-β1, Smad3 or Ctgf ASOs resulted in significant improvement in tendon gliding function at 14 and 21 days, relative to control. Consistent with a decrease in adhesions, Col3a1 expression was significantly decreased in Tgf-β1, Smad3 and Ctgf ASO treated tendons relative to control. Smad3 ASO treatment enhanced the max load at failure of healing tendons at 14 days, relative to control. Taken together, these data support the use of ASO treatment to improve FT repair, and suggest that modulation of the Tgf-β1 signaling pathway can reduce adhesions while maintaining the strength of the repair. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
A versatile and robust microfluidic nanoprecipitation platform for high throughput synthesis of nanoparticles is fabricated. The versatility of this platform is proven through the successful preparation of different type of nanoparticles. This platform presents great robustness, with homogeneous nanoparticles always being obtained, regardless of the formulation parameters. The diameter and surface charge of the prepared nanoparticles can also be easily tuned.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Flexor tendon injuries caused by deep lacerations to the hands are a challenging problem as they often result in debilitating adhesions that prevent the movement of the afflicted fingers. Evidence exists that tendon adhesions as well as scarring throughout the body are largely precipitated by the pleiotropic growth factor, TGF-β1, but the effects of TGF-β1 are poorly understood in tendon healing. Using an in vitro model of tendon healing, we previously found that TGF-β1 causes gene expression changes in tenocytes that are consistent with scar tissue and adhesion formation, including upregulation of the anti-fibrinolytic protein, PAI-1. Therefore, we hypothesized that TGF-β1 contributes to scarring and adhesions by reducing the activity of proteases responsible for ECM degradation and remodeling, such as plasmin and MMPs, via upregulation of PAI-1. To test our hypothesis, we examined the effects of TGF-β1 on the protease activity of tendon cells. We found that flexor tendon tenocytes treated with TGF-β1 had significantly reduced levels of active MMP-2 and plasmin. Interestingly, the effects of TGF-β1 on protease activity were completely abolished in tendon cells from homozygous PAI-1 KO mice, which are unable to express PAI-1. Our findings support the hypothesis that TGF-β1 induces PAI-1, which suppresses plasmin and plasmin-mediated MMP activity, and provide evidence that PAI-1 may be a novel therapeutic target for preventing adhesions and promoting a scarless, regenerative repair of flexor tendon injuries. © 2014 Wiley Periodicals, Inc.