Immuno-engineering of Cells and Biomaterials for Biomedical Applications

Immuunijärjestelmän muodostavat immuunisolut, jotka pitävät kehomme turvassa ja auttavat torjumaan eri taudinaiheuttajien välittämiä infektioita sekä ovat keskeisessä roolissa homeostaasin säätelyssä, haavan paranemisessa ja kudosten uusiutumisessa. Onnistuneen hoidon saavuttamiseksi on potilaan kehoon lisätyn implantin tai muiden vieraiden aineiden, kuten biomateriaalien, nanopartikkeleiden, pienimolekyylisten lääkkeiden tai elävien solujen selviydyttävä monimutkaisessa immuuniympäristössä. Tämän vuoksi on ensiarvoisen tärkeää kehittää uusia teknisiä ratkaisuja tai immuunivasteisia biomateriaaleja, jotka pystyvät turvallisesti muuttamaan aggressiivista immuunijärjestelmää tai hyödyntämään immuunijärjestelmän "soluja tappavia" tai "haavoja parantavia" ominaisuuksia potilaiden sairauksien hoidossa. Tähän ongelmaan liittyen tässä väitöskirjassa kehitettiin uusia biotekniikan strategioita, jotka hyödyntävät nanolääketieteen tai kudosteknologian lähestymistapoja immuunijärjestelmän muuttamiseksi. Tässä väitöskirjassa on käytetty nanokantajien ja hydrogeelitukirakenteiden suunnittelussa hyaluronihappoa (eng. hyaluronic acid, HA). HA-pohjaiset nanopartikkelit suunniteltiin syövän vastaisiin sovelluksiin, kun taas immunosuppressiiviset HA-pohjaiset hydrogeelitukirankenteet suunniteltiin kudosteknologian sovelluksiin. HA-nanopartikkelit suunniteltiin käyttäen uutta itsekokoontumisstrategiaa, jossa vettä hylkivä immunosuppressiivinen lääke, deksametasoni, yhdistettiin HA-runkoon amfifiilisyyden luomiseksi. Tämän jälkeen tunnettu kemoterapialääke, doksorubisiini, lastattiin nanopartikkeliin. Nämä lääkettä sisältävät HA-nanopartikkelit muodostivat ihanteellisen nanokantajan yhdistelmähoitoon ja lääkkeenkuljetusjärjestelmää tutkittiin sen syövänvastaisen aktiivisuuden, makrofagien polarisaatiopotentiaalin ja sen kyvyn muuttaa trombiintulehdusta ihmisen kokoveressä. Havaittiin, että uusi HA-pohjainen nanokantaja esti verihiutaleiden yhteen kasautumista ja trombiinin muodostumista, mitä doksorubisiini yleensä aiheuttaa. HA-pohjainen nanokantaja aiheutti myös kasvaimeen liittyvien makrofagien repolarisaation M1-makrofageiksi, mikä on osoituksena tulehdusta edistävien geenien, kuten TNF:n, ilmentymisen lisääntymisestä. Toinen väistökirjan keskeinen tavoite on terapeuttisten solujen, kuten mesenkymaalisten kantasolujen (eng. mesenchymal stem cells, MSC) toimittaminen kohteeseen. MSC-solujen aiheuttama tromboottinen aktiivisuus allotransplantaation jälkeen on suuri haaste solupohjaisissa hoidoissa, koska nämä solut laukaisevat veren hyytymisen ja komplementtijärjestelmän aktivoitumisen suonensisäisen infuusion jälkeen. Tämä johtaa solujen huonoon in vivo eloonjäämiseen. Haasteen välttämiseksi hyödynsimme kahta riippumatonta strategiaa. Suunnittelimme uudet pluronic- pohjaiset nanomisellit, jotka oli yhdistetty pelkistävää disulfidilinkkeria sisältävään siRNA:han. Tämä yhdistelmä osoittautui tehokkaaksi geenin vaimentamisessa (~ 70 %) MSC-soluissa. Kudostekijägeenin tehokas vaimentaminen MSC-soluissa paransi merkittävästi veriyhteensopivuutta ihmisen kokoveressä, koska ne eivät aktivoineet komplementti- ja hyytymisreittejä. Yllättäen kudostekijän vaimentaminen johti parakriinisen signaalin ja erilaistumispotentiaalin parantumiseen ennestään tuntemattoman molekyylireitin kautta. Toisessa strategiassa pinnoitimme MSC-solut käyttäen kahta polyelektrolyytiä layer-by-layer (LbL)-tekniikalla: hepariinia ja gelatiinia (H/G). Näiden päällystettyjen solujen havaittiin parantavan veriyhteensopivuutta, kun niitä tutkittiin ihmisen kokoveressä. Myös näiden päällystettyjen solujen lisääntyminen, parakriininen signalointi ja erilaistuminen tutkittiin. Tutkimuksista paljastui, että H/G-pinnoite ei estänyt solujen parakriinisia signalointi- ja erilaistumiskykyjä. Lopuksi kehitimme immuunivasteisen HA-hydrogeelin konjugoimalla gallushappoa. Gallolin lisääminen HA-hydrogeeliin paransi adhesiivisia sekä antioksidanttisia ominaisuuksia, samalla kun se tarjosi hydrogeelille pseudoplastiset/itseparantuvat ominaisuudet. Yllättäen gallushapon funktionalisointi tarjosi immunosuppressiivisia ominaisuuksia, mikä varmistettiin viljelemällä primäärisiä hiiren M1-makrofageja. Gallolifunktionalisoitujen HA- geelien ihonalainen implantointi terveisiin hiiriin osoitti immunosuppressiivisten myeloidisolujen tunkeutumista näissä hydrogeeleissä. Kun taas kontrolli-HA- geeleissä havaittiin suurempi määrä tulehdusta edistäviä myeloidisoluja. Yhteenvetona voidaan todeta, että tämä väitöskirja esittelee laajan valikoiman teknisiä strategioita immuunijärjestelmän säätelyyn. Strategiat tukivat kantasolujen parempaa eloonjäämistä transplantaation jälkeen, tarjosivat kemoterapeuttisten aineiden turvallisemman kuljettamisen käyttämällä biomimeettisiä HA-pohjaisia nanokantajia ja tunnistivat gallushapon immunomodulatoriseksi molekyyliksi, jota voidaan hyödyntään immunosuppressiivisten 3D tukirakenteiden suunnittelussa kudosteknologian sovelluksiin.Immune cells that constitute our immune system keep the human body safe and help in warding off infections mediated by different pathogens and also play a key role in regulating homeostasis, wound healing, and regeneration of tissues. Implantation or infusion of foreign agents such as biomaterials, nanoparticles, small molecule drugs, or living cells needs to navigate the complex immune landscape for successful therapeutic outcomes. Thus, devising new engineering tools or immune-responsive biomaterials that can safely traverse/modulate the aggressive immune system or harness the ‘cell-killing’ or ‘wound healing’ properties of the immune system as a potent tool for treating human diseases are of paramount importance for developing new therapies. In this context, this dissertation has developed new bioengineering strategies adopting nanomedicine or tissue engineering approaches for modulating our immune system which was successfully demonstrated for anti-cancer studies, stem cell delivery, and tissue regeneration applications. Here in this thesis, hyaluronic acid (HA), one of the key polymers of our extracellular matrix (ECM) has been used as a major building block for engineering nanocarriers and a hydrogel scaffold. The HA-derived nanoparticles were designed for anti-cancer applications and immunosuppressive HA-based hydrogel scaffolds were designed for tissue engineering applications. The HA nanoparticles were designed following a novel self-assembly strategy where hydrophobic immunosuppressive drug, dexamethasone was conjugated to the HA backbone to induce amphiphilicity, which was subsequently loaded with a well-known chemotherapy drug, doxorubicin. These drug-loaded HA-nanoparticles formed an ideal nanocarrier for combination therapy and the delivery system was tested for its anticancer activity, macrophage polarization potential, and its capability to modulate thromboinflammation in human whole blood. Gratifyingly, the novel HA-based nanocarrier was found to suppress platelet aggregation and thrombin generation that are trigged by doxorubicin. The HA-based nanocarrier also triggered the repolarization of tumor-associated macrophages to M1 macrophages as evidenced by the increase in expression of proinflammatory genes like TNF. Delivering therapeutic cells such as mesenchymal stem cells (MSCs) is another key objective of this thesis. The thrombotic activity induced by MSCs after allotransplantation is a major obstacle in the field of cell-based therapies as these cells trigger the activation of coagulation and complement cascade, after intravenous infusion leading to poor in-vivo survival. To circumvent this challenge, we adopted two independent approaches, namely nanoparticle-mediated tissue factor gene silencing and layer-by-layer polyelectrolyte coating. We designed a novel pluronic- based nanomicelles conjugated with siRNA bearing a reducible disulfide linker that displayed efficient gene silencing (~70%) in MSCs. The effective silencing of the tissue factor gene in MSCs significantly enhanced the hemocompatibility in human whole blood as they did not activate the complement and coagulation pathways. Surprisingly, the silencing of tissue factor resulted in enhanced paracrine signaling and differentiation potential through some unknown molecular pathway. In the second strategy, we coated the MSCs with two polyelectrolytes namely, heparin and gelatin (H/G) in a layer by layer (LbL) manner. These coated cells were found to enhance hemocompatible when studied in human whole blood. The proliferation, paracrine signaling aspects and differentiation aspects of these coated cells were also studied. These studies revealed that the H/G coating did not hinder the paracrine signaling and differentiation capabilities of the cells. Finally, we engineered an immunoresponsive HA hydrogel by conjugating gallic acid, a polyphenol that is known to display antioxidant properties and are found in green tea, and other medicinal plants. The addition of gallol to HA hydrogel enhanced the adhesive and antioxidant properties while also providing a shear thinning/self-healing property to the hydrogel. Surprisingly, gallic acid functionalization provided immunosuppressive characteristics as verified by culturing primary murine M1 macrophages. Subcutaneous implantation of gallol- functionalized HA gels in healthy mice displayed infiltration of immunosuppressive myeloid cells within these gels, while higher numbers of pro-inflammatory myeloid cells were observed in the control HA gels. In conclusion, this dissertation formulates a wide range of engineering strategies for regulating our immune system that supported enhanced survival of stem cells after transplantation, provided safer delivery of chemotherapeutic agents using biomimetic HA-based nanocarriers, and identified gallic acid as an immunomodulatory molecule that can be exploited to design immunosuppressive 3D scaffolds for tissue engineering applications

Predictors of hospital stay and mortality in dengue virus infection-experience from Aga Khan University Hospital Pakistan

Background: Dengue virus infection (DVI) is very common infection. There is scarcity of data on factor associated with increased hospitalstay and mortality in dengue virus infection (DVI). This study was done to know about factors associated with increased hospital stay and mortality in patients admitted with DVI. Results: Out of 532 patients, two third (72.6%) had stay ≤3 days while one third (27.4%) had stay greater than 3 days. The mean length of hospital stay was 3.46 ± 3.45 days. Factors associated with increased hospital stay (\u3e3 days) included AKI (acute kidney injury) (Odd ratio 2.98; 95% CI 1.66-5.34), prolonged prothrombin time (Odd ratio 2.03; 95% CI 1.07-3.84), prolonged activated partial thromboplastin time (aPTT) (Odd ratio 1.80; CI 95% 1.15-2.83) and increased age of \u3e 41.10 years (Odd ratio 1.03; CI 95% 1.01-1.04).Mortality was 1.5%. High mortality was found in those with AKI (P \u3c0.01), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (P \u3c0.001), respiratory failure (P0.01), prolong PT (P 0.001), prolong aPTT (P0.01) and increased hospital stay (P0.04). Conclusion: Increasing age, coagulopathy and acute kidney injury in patients with DVI is associated with increased hospital stay. Morality was more in patients with AKI, DHF and DSS, respiratory failure, coagulopathy and these patients had more prolonged hospitalization

A rare case of Aeromonas hydrophila catheter related sepsis in a patient with chronic kidney disease receiving steroids and dialysis: a case report and review of Aeromonas infections in chronic kidney disease patients

Aeromonas hydrophila (AH) is an aquatic bacterium. We present a case of fifty-five-year-old gentleman with chronic kidney disease (CKD) due to crescentic IgA nephropathy who presented to us with fever. He was recently pulsed with methyl prednisolone followed by oral prednisolone and discharged on maintenance dialysis through a double lumen dialysis catheter. Blood culture from peripheral vein and double lumen dialysis catheter grew AH. We speculate low immunity due to steroids and uremia along with touch contamination of dialysis catheter by the patient or dialysis nurse could have led to this rare infection. Dialysis catheter related infection by AH is rare. We present our case here and take the opportunity to give a brief review of AH infections in CKD patients

Hyaluronan derived nanoparticle for simvastatin delivery: evaluation of simvastatin induced myotoxicity in tissue engineered skeletal muscle

Statins are currently the most prescribed hypercholesterolemia-lowering drugs worldwide, with estimated usage approaching one-sixth of the population. However, statins are known to cause pleiotropic skeletal myopathies in 1.5% to 10% of patients and the mechanisms by which statins induce this response, are not fully understood. In this study, a 3D collagen-based tissue-engineered skeletal muscle construct is utilised as a screening platform to test the efficacy and toxicity of a new delivery system. A hyaluronic acid derived nanoparticle loaded with simvastatin (HA-SIM-NPs) is designed and the effect of free simvastatin and HA-SIM-NPs on cellular, molecular and tissue response is investigated. Morphological ablation of myotubes and lack of de novo myotube formation (regeneration) was evident at the highest concentrations (333.33 μM), independent of delivery vehicle (SIM or HA-SIM-NP). A dose-dependent disruption of the cytoskeleton, reductions in metabolic activity and tissue engineered (TE) construct tissue relaxation was evident in the free drug condition (SIM, 3.33 μM and 33.33 nM). However, most of these changes were ameliorated when SIM was delivered via HA-SIM-NPs. Significantly, homogeneous expressions of MMP2, MMP9, and myogenin in HA-SIM-NPs outlined enhanced regenerative responses compared to SIM. Together, these results outline statin delivery via HA-SIM-NP as an effective delivery mechanism to inhibit deleterious myotoxic side-effects

Imaging spectrum of extrapulmonary manifestations of COVID-19 infection- a multi-centre descriptive study from Southern India

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is the causative agent for coronavirus disease 2019 (COVID-19) pandemic. While the primary organ of involvement in this disease is the lung, multiple other organ systems can be involved either due to direct viral cytopathic effects or due to thrombo-inflammation and immune system dysregulation. In this study we describe the spectrum of extrapulmonary imaging findings encountered in our patients with COVID-19.Methods: This was a retrospective observational study conducted in three tertiary care hospitals in the city of Chennai in southern India. All cross-sectional imaging studies (other than lung imaging studies) performed in patients who had proven COVID-19 infection by RT-PCR testing during the period from April 2020 to March 2021 were included as part of the study. Extrapulmonary findings in these imaging studies were recorded and collated system-wise.Results: A total of 96 non-lung imaging studies were performed in patients who had RT-PCR positivity for COVID-19 infection. Among these a total of 30 studies had extrapulmonary imaging findings. Vascular involvement was seen in 14 patients, central nervous system involvement in 13 patients, abdomen involvement in 2 patients, and cardiac involvement in 1 patient. Vascular manifestations included arterial and venous thrombosis. Neurological manifestations included stroke, encephalitis and demyelination. Abdominal manifestations included enteritis and acute kidney injury. Cardiac manifestation was in form of myocarditis.Conclusions: Extrapulmonary imaging findings in COVID-19 are uncommon but not rare. Multisystem thrombotic manifestations and central nervous system involvement account for majority of extrapulmonary imaging findings in COVID-19

Heparin-Derived Theranostic Nanoprobes Overcome the Blood-Brain Barrier and Target Glioma in Murine Model

The poor permeability of theranostic agents across the blood-brain barrier (BBB) significantly hampers the development of new treatment modalities for neurological diseases. A new biomimetic nanocarrier is discovered using heparin (HP) that effectively passes the BBB and targets glioblastoma. Specifically, HP-coated gold nanoparticles (HP-AuNPs) are designed that are labeled with three different imaging modalities namely, fluorescein (FITC-HP-AuNP), radioisotope (68)Gallium (Ga-68-HP-AuNPs), and MRI active gadolinium (Gd-HP-AuNPs). The systemic infusion of FITC-HP-AuNPs in three different mouse strains (C57BL/6JRj, FVB, and NMRI-nude) displays excellent penetration and reveals uniform distribution of fluorescent particles in the brain parenchyma (69-86%) with some accumulation in neurons (8-18%) and microglia (4-10%). Tail-vein administration of radiolabeled Ga-68-HP-AuNPs in healthy rats also show Ga-68-HP-AuNP inside the brain parenchyma and in areas containing cerebrospinal fluid, such as the lateral ventricles, the cerebellum, and brain stem. Finally, tail-vein administration of Gd-HP-AuNPs (that displays approximate to threefold higher relaxivity than that of commercial Gd-DTPA) in an orthotopic glioblastoma (U87MG xenograft) model in nude mice demonstrates enrichment of T1-contrast at the intracranial tumor with a gradual increase in the contrast in the tumor region between 1 and 3 h. It is believed, the finding offers the untapped potential of HP-derived-NPs to deliver cargo molecules for treating neurological disorders.Peer reviewe

Hyaluronic acid based next generation bioink for 3D bioprinting of human stem cell derived corneal stromal model with innervation

Corneal transplantation remains gold standard for the treatment of severe cornea diseases, however, scarcity of donor cornea is a serious bottleneck. 3D bioprinting holds tremendous potential for cornea tissue engineering (TE). One of the key technological challenges is to design bioink compositions with ideal printability and cytocompatibility. Photo-crosslinking and ionic crosslinking are often used for the stabilization of 3D bioprinted structures, which can possess limitations on biological functionality of the printed cells. Here, we developed a hyaluronic acid-based dopamine containing bioink using hydrazone crosslinking chemistry for the 3D bioprinting of corneal equivalents. First, the shear thinning property, viscosity, and mechanical stability of the bioink were optimized before extrusion-based 3D bioprinting for the shape fidelity and self-healing property characterizations. Subsequently, human adipose stem cells (hASCs) and hASC-derived corneal stromal keratocytes were used for bioprinting corneal stroma structures and their cell viability, proliferation, microstructure and expression of key proteins (lumican, vimentin, connexin 43,α-smooth muscle actin) were evaluated. Moreover, 3D bioprinted stromal structures were implanted intoex vivoporcine cornea to explore tissue integration. Finally, human pluripotent stem cell derived neurons (hPSC-neurons), were 3D bioprinted to the periphery of the corneal structures to analyze innervation. The bioink showed excellent shear thinning property, viscosity, printability, shape fidelity and self-healing properties with high cytocompatibility. Cells in the printed structures displayed good tissue formation and 3D bioprinted cornea structures demonstrated excellentex vivointegration to host tissue as well asin vitroinnervation. The developed bioink and the printed cornea stromal equivalents hold great potential for cornea TE applications.publishedVersionPeer reviewe