Bio-imaging And Photodynamic Therapy Effect Of Tetra Sulphonatophenyl Porphyrin （TSPP） With TiO₂ Nanowhiskers For Rheumatoid Arthritis Theranostics

Biocompatibility and toxicity evaluation of Tetra Sulphonatophenyl Porphyrin and TiO2 nanocompositesTetra Sulphonatophenyl Porphyrin (TSPP) is well known photosensitizer for photodynamic therapy; nevertheless, its well-known adverse effects hamper its potential use. Recently, nano TiO2’s potential role in biomedical has been defined for various disease theranostics, including cancer and other infections. Thus, in this contribution we have explored the possibility of utilizing TiO2 nanowhiskers as novel strategy to lower TSPP adverse effects both in vitro, and in vivo. Various concentrations of TSPP, TiO2-TSPP, and TiO2 were injected to three different rat groups, while fourth group was kept as control. Toxic effects were evaluated on excretory and circulatory system by using histopathology, fluorescent microscopy, complete blood cells count (CBC) and serum enzymes. In complete blood cells count, all cells were significantly (p< 0.01) affected by the various concentration and treatment groups. The various dose concentrations and treatment also significantly (p< 0.01) affected the serum enzyme parameters including AST, ALT, LDH, Creatinine and BUN level. The low concentration of TSPP-TiO2 was found to be safest, on the bases of serum enzyme parameters, CBC, histopathology, and fluorescent microscopic analysis. The MTT assay was used to evaluate in vitro cytotoxicity, and the results demonstrated maximum viability in illuminated TSPP-TiO2 nanowhiskers group when compared with TSPP treated group. It was evident that increase in concentration of TSPP increased the toxic effects; however, the TiO2 nanowhiskers combination with TSPP decreased these adverse effects. Moreover, TSPP (0.1 mM) combined with TiO2 nanowhiskers (0.6 mM) was safer than TSPP (0.1 mM) alone.Rheumatoid arthritis theranostics by photoactivated TSPP-TiO2 nanocompositesRheumatoid Arthritis (RA) is one of the major human progressive joint inflammatory diseases affecting one percent of population. It is considered to be an autoimmune disease with multiple triggers. To date, early diagnosis and treatment of RA remains a major challenge. Recently, nano titanium dioxide (TiO2) potential role in biomedical has been defined for various disease theranostics, including cancer and other infections. In this contribution we have explored the possibility to utilize novel nanocomposites of tetra sulphonatophenyl porphyrin (TSPP) with TiO2 nanowhiskers (TP) as effective bioimaging and photodynamic therapeutic (PDT) agent for RA theranostics. Our observations demonstrate that TP solution PDT have an ameliorating effect on the RA by decreasing significantly (p< 0.01) the IL-17 and TNF-a level in blood serum. Besides, the fluorescent bioimaging enabled us to diagnose the disease in subclinical stages before the onset of clinical signs and bio-mark the RA insulted joint.Rheumatoid arthritis stress biomarkers evaluation during photodynamic therapy with nanocomposites TSPP-TiO2 and its influence on Bone Marrow Stem Cells in vitroPhotodynamic therapy (PDT) is mostly used to induce apoptosis or necrosis in the benign and malignant tumors, along with other microbial infections and suppression of autoimmune diseases including Rheumatoid Arthritis (RA). In this study we used Tetra Sulphonatophenyl Porphyrin (TSPP) with TiO2 nanowhiskers for RA PDT and evaluated their effect on stress bio-markers (i.e., CAT, SOD, GPX, GR, TAO and MDA) in vivo and BMSC proliferation in vitro. We compared four murine groups, three of which had Collagen Induced Arthritis as TP-L (illuminated), TP-nL (dark) and CIA (control), whereas the other group was normal without disease and treatment. All anti-oxidative enzymes and biomarkers were significantly (p< 0.01) affected by the treatment, except TAO (p> 0.05). Moreover, we also evaluated the growth proliferating effect of TSPP-TiO2 (TP) PDT on the in vitro RA infected BMSC, i.e.,25μl TP had highest cell count (12.33x106 cells/well) and 33% more growth rate in photoactivated TP when compared with 50 and 100 μl TP treatment groups. The bone marrow stem cells (BMSC) are also a focus in translational medicine, tissue engineering and as an autoimmune disease suppressant. Therefore, we co-cultured the RA synovial fibroblast cells as normal BMSC to check their effect on the inflammatory cytokines secretion (TNF-α and IL-17). The results demonstrated that BMSC significantly (p< 0.01) lowered the TNF-α secretion (from 31.10±1.92 (SD) to 16.12±1.45 (SD) ng L-1) in the RA synovial fibroblast cells, whereas IL-17 remained un-affected. We found that photoactivated TSPP-TiO2 nanocomposites for RA PDT may be safer than photosensitizers without the titanium nanomaterials in terms of reduced oxidative stress and also promotion of RA BMS cells growth in vitro as novel finding, whereas BMSC lower the RA biomarkers from fibroblast cells.Synergistic effect of allogenic BMSC and TSPP-TiO2 nanocomposites based PDT on RA ameliorationRA etiology and amelioration is a challenge to modern therapeutics. In spite of skeleton supporting role, the BMSC has been reported for having ameliorating effect on various auto immune diseases. Similarly, the theranostic properties of TSPP-TiO2 nanocomposites encouraged its further utility in biomedical realms. Therefore, the synergistic effect of allogenic BMSC and TSPP-TiO2 nanocomposites could revolutionize RA theranostics. We found that allogenic BMSC combined with TSPP-TiO2 nanocomposites can significantly (p< 0.01) lower the CIA murine model serum biomarkers (i.e., TNF-α and IL-17) both in vitro and in vivo, along with other parameters, i.e., arthritis score, BMSC count, CBC, PLT, RBC, WBC. Moreover, the fluorescence of the diseased sites on feet, long bones and X-ray bioimaging of RA joints revealed clinical efficacy of BMSC combined with TSPP-TiO2 nanocomposites. We are optimistic that the aforementioned findings will provide new approach in RA theranostics.RA BMSC translation via miRNAs during post PDT ex vivo evaluationThe post-translational genes regulation by microRNAs (miRNA) has been extensively investigated for various diseases, notably in cancer, cardiovascular diseases and auto-immune maladies e.g. RA. The BMSC share same niche with hematopoietic stem cells, adipocytes and osteocytes within bone marrow. Therefore, their altered functionality will influence the adjacent cells performance. Moreover, it is noted that in RA, the bilateral insult in bone tissue increases the chance of effect on various cells functionality. After PDT together with TSPP and TSPP-TiO2 nanocomposites the RA BMSC were evaluated for miRNAs profile. We found that rno-mir-375-3p and rno-mir-196b-3p were 100 times up regulated (100.97 and 100.22, respectively), as compared to control. Similarly, rno-mir-rno-miR-143-5p (34.40), rno-miR-141-3p (32.21), rno-miR-206-3p (21.82), rno-miR-181b-5p (21.45), rno-miR-190a-5p (21.17), rno-miR-301a-3p (14.07), rno-miR-497-5p (11.62) and rno-miR-363-5p (10.88) has been included in top ten up regulated miRNAs in TSPP-TiO2 nanocomposites treated group BMSC. Since, the TSPP-TiO2 nanocomposites treatment had excellent ameliorating effect on the CIA murine models as compared to that of TSPP alone and control group BMSC; hence we are optimistic that these up-regulated miRNAs may definitely have regulatory effect on the adjacent hematopoietic stem cells and synovial joint milieu.The overall results suggest that when TSPP combined with TiO2 nanowhiskers, the relevant toxic effects are mitigated and its biocompatibility improved. Similarly, we have not only achieved the early diagnosis of RA but also successfully ameliorated the disease, showing TP effects in theranostics. The TP biomedical application can not only be confined to the RA but can also be extended to other infectious and non-infectious diseases, especially the diabetes mellitus type II as our recent additional study.Extension of Photoactivated TiO2 Nanowhiskers and Tetra Sulphonatophenyl Porphyrin therapeutic potential to other disease like Diabetes MellitusDiabetes Mellitus (DM) is one of the major human diseases resulting in hyperglycemia. Photodynamic therapy (PDT) is well-known for ablation of neoplastic tissues and amelioration of autoimmune diseases. Therefore, we hypothesized the normoglycemic effect of TSPP-TiO2 PDT on the DM (type Ⅰ and Ⅱ). We used three groups of DM murine models, i.e., type I, type II and normal group. These results demonstrate that photoactivated TSPP-TiO2 nanocomposites had significantly (p< 0.01) lowered the blood glucose level in type II DM group, whereas the type I DM group remained non-significant. Moreover, evaluation of the blood sugar level in various time intervals (1,2,8 and 24 hours) post PDT revealed 31 and 33% decline after one and two hours respectively, and then incline was noticed. These results suggest the normoglycemic effect of photoactivated TSPP-TiO2 nanocomposites on type II DM that can be used for disease amelioration, subject to further investigations.