DESC:SYNTHESIS OF MESO-TRITOLYL-BF2-OXASMARAGDYRIN BASED QUANTUM DOTS

FIELD OF THE INVENTION
[0001] The present invention generally relates to a process for synthesizing agents for bio-imaging and photothermal therapy, and more particularly relates to a process for synthesizing meso-Tritolyl-BF2-Oxasmaragdyrin based nanoparticles (TBSNPs).

BACKGROUND OF THE INVENTION
[0002] A rapid increase in cancer and associated mortalities in recent years promoted cancer to be a leading cause for mortalities, second only to non-communicable diseases. Current cancer therapeutic modalities are limited to surgery, chemotherapy, radiotherapy, and immunotherapy. Due to the heterogeneity of cancers, these therapeutic approaches are often limited to specific types, and this coupled with their associated side effects motivated researchers to search for alternative approaches. Photothermal therapy (PTT) is an emerging therapeutic strategy that offers minimal invasiveness, ease of operation, rapid treatment and quick patient recovery. In the recent years various photothermal agents have been screened for PTT applications, and a few of them are gold nanoparticles, graphene and graphene oxide, CNTs, Cdots, black phosphorus, palladium, copper sulfide, copper selenide, molybdenum oxide, molybdeniumdisulfide, magnetic nanoparticles and polymers such as polyanaline, polypyrroles. The non-biodegradability of inorganic nanoparticles, coupled with a possibility of long-term toxicity is a serious setback for its in vivo applications.
[0003] To alleviate these problems, a biodegradable and an efficient photothermal agent needs to be developed and organic dyes could be an ideal candidate. The dyes possessing absorption/fluorescence in Near Infra-red region (650-950 nm and 1100-1300 nm) is especially favorable for Near Infra-red Fluorescence (NIRF) imaging and/or therapeutic applications due to low absorption and scattering in mammalian cells resulting in higher penetration and better signal to noise ratio. Hence there is a growing requirement to develop dyes that absorb and emit strongly in the NIR region. To date, there are a limited number of dyes which fluoresce in this region such as cyanines, phthalocyanines, squaraine derivatives, BODIPY/Aza BODIPY dyes, xanthenes, and a small selection of porphyrin derivatives.
[0004] One such molecule which belongs to a class of expanded porphyrins that absorb and emit in the NIR-I region is Smaragdyrin. Smaragdyins are aromatic expanded pentapyrrolic macrocycles with five pyrroles connected by three meso carbons and two direct pyrrole-pyrrole bonds. Figure 16 of the present invention illustrates chemical structures known in the art which exhibit NIR fluorescence and which can be employed in Photothermal therapy. Chandrasekhar and coworkers reported the synthesis of a stable meso-triaryl-25-oxasmaragdyrin which is an analog of smaragdyrin in which one of the pyrrole rings is replaced with furan. The 25-oxasmaragdyrins absorb and emit in the Vis-NIR region with moderate quantum yields and singlet state lifetimes. The photophysical properties were further improved by synthesizing BF2- and PO2- complexes of 25-oxasmaragdyrin. Though these molecules have good photophysical properties, their potential for biological applications was not explored due to their poor physicochemical properties and aqueous solubility. A perusal of literature revealed that various dye-based formulations had been reported for their use as NIRF imaging as well as photothermal therapeutic applications.

SUMMARY OF THE INVENTION
[0005] This summary is provided to introduce a selection of concepts in a simplified format that are further described in the detailed description of the invention. This summary is not intended to identify key or essential inventive concepts of the claimed subject matter, nor is it intended for determining the scope of the claimed subject matter.
[0006] The present invention discloses a process to synthesize highly photostable and biocompatible Meso-tritolyl-BF2-oxasmaragdyrin (TBS) based Nanoparticles/Quantum dots (TBS-QDs). The process comprises synthesizing meso-Tritolyl-BF2-Oxasmaragdyrin using a method reported in M. Rajeswara Rao, M. Ravikanth, J. Org. Chem. 2011, 76, 3582-3587; preparing a mixture of TBS and lipids in an organic solvent; forming a thin film of said mixture by evaporating the organic solvent; hydrating the film with water; forming a dispersion by homogenizing the hydrated film at high pressure to obtain a solution of TBS-QDs and characterizing the obtained TBS-QD solution.

BRIEF DESCRIPTION OF THE DRAWINGS
[0007] Reference will be made to embodiments of the invention, examples of which may be illustrated in the accompanying figures. These figures are intended to be illustrative, not limiting. Although the invention is generally described in the context of these embodiments, it should be understood that it is not intended to limit the scope of the invention to these particular embodiments.
[0008] The objects, features, and advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
[0009] Figure 1 illustrates synthesis of TBSNPs.
[00010] Figure 2 is a photographic and graphical representation ofCharacterisation of TBSQDs (a) Comparison of Raman scattering for TBS and TBSQDs on Ag nanoparticles. (b-d) TEM Images [(b) HRTEM of TBSQDs (Inset shows FFT) (c) TEM image of TBSQDs (inset shows the Selected Area Electron Diffraction (SAED)].
[00011] Figure 3 is a graphical representation of Particle size distribution of TBS-QDs calculated by TEM.
[00012] Table 1 illustrates Full width half maximas of selected Surface enhanced raman signals.
[00013] Figure 4 is a graphical representation of Photophysical properties. Comparison of (a) Normalized Absorption, (b) Normalized fluorescence, and (c) Fluorescence decay profile of TBSQDs with TBS where 440 nm is used for excitation. 3D heat map for transient absorption spectra of TBS (d) and TBSQDs (h) excited at 400 nm. Transient absorption spectra at different delays for TBS (e) and TBSQDs (i). Kinetic decay fit of TBS probed at 440 nm (f), 469 nm (g) and TBSQDs probed at 440 nm (j), 469 nm (k), 712 nm (l), 724 nm (m).
[00014] Figure 5 is a graphical representation of absorption spectra of TBS and TBSQDs. (0.05mg/mL concentration)
[00015] Table 2 illustrates absorption and fluorescence data for TBS and TBSQDs.
[00016] Figure 6 is a graphical representation of absorption, emission and excitation spectra of TBS in CHCl3.
[00017] Figure 7 is a graphical representation of absorption, emission and excitation spectra of TBSQDs in H2O.
[00018] Table 3 illustrates transient absorption decay lifetimes (ps) with respective contributions probed at different wavelengths.
[00019] Table 4 illustrates sequential model sum of squares.
[00020] Table 5 illustrates ANOVA for the response, temperature for quadratic model.
[00021] Figure 8 is a graphical representation of Photo-thermal transduction efficiency (a) 3D plot, (b) design space for the response; temperature, (c) temperature change corresponding to laser ON and OFF of TBSQDs, (d) time vs. ln (?) from the cooling stage of TBSQDs, (e) temperature vs. time profile of Milli-Q, and (f) multi-cycle temperature changes upon exposure of TBSQDs with 750 nm laser.
[00022] Table 6 illustrates confirmatory trials to evaluate the design space
[00023] Figure 9 is a photographic and graphical representation of Biocompatibility and Hemocompatibility Studies (Top to bottom) (a) Graph showing the biocompatibility of TBSQDs in L929 cells at various concentrations, (b) Graph showing the percentage hemolysis for the hemocompatibility study of TBSQDs, (c) Digital image of hemocompatibility assay of TBSQDs with positive and negative control, and ESEM images were also captured for analysing the morphology with (d) showing negative control,(e) showing positive control, (f) showing samples treated with TBSQDs, which have morphology alike to the negative control.
[00024] Figure 10 is a graphical representation of In vitro cellular uptake and photo-thermal efficacy (a) Uptake of TBSQDs in 4T1 cells, (b) graph depicting in vitro photo-thermal efficacy in 4T1 cells, (c) FACS scatter plot of Annexin-V PI staining in control and treated cells.
[00025] Figure 11 is a photographic representation of CLSM images showing 4T1 Control cells treated with DAPI (a) Differential Interference Contrast (DIC) image, (b) DAPI channel with 410-454 nm emission filter, (c) Fluorescence channel with 680-730 nm emission filter, (d) merged image of all channels and 4T1 cells treated with DAPI and TBSQD, (e) Differential Interference Contrast (DIC) image, (f) DAPI channel with 410-454 nm emission filter, (g) Fluorescence channel with 680-730 nm emission filter and (h) merged image of all channels.
[00026] Figure 12 is a graphical and photographic representation of whole body fluorescence imaging and tumor homing (a) Whole body fluorescence imaging of the control and TBSQDs injected mice (b) Quantification of total fluorescence from the whole body of control and TBSQDs injected mice (c) Fold change in fluorescence output in TBSQDs injected as compared to the control mice (d) Fluorescence images showing fractional tumor homing of the formulation in TBSQDs injected mice (e) Absolute quantification of the amount of TBSQDs in the tumor tissue of control and TBSQDs injected mice.
[00027] Figure 13 is a graphical and photographic representation of Biodistribution and single dose toxicity (a-b) Ex vivo fluorescence imaging of vital organs; spleen (S), lungs (Lu), liver (Li), kidney (Ki) and heart (H) collected from control and TBSQDs injected mice. (c) Qualitative evaluation of fluorescence output from organ samples of control and TBSQDs injected mice. (d) Quantification of the amount of TBSQDs in organ samples collected from control and TBSQDs injected mice. Toxicity evaluation of TBSQDs in preclinical settings (e-f) Hematoxylin and Eosin staining of the organ samples of heart, kidney, liver, spleen and lung sections of control and TBSQDs injected mice. (g) Serum biochemical analysis of alanine transaminase (ALT), creatinine (CRE), urea (BUN), and aspartate aminotransferase (AST) in control and TBSQDs injected mice.
[00028] Figure 14 is a graphical and photographic representation of In vivo photo-thermal ablation by TBSQDs in a 4T1 xenograft mouse model: (a) Representative in vivo bioluminescence images of mice from various treatment groups, (b) Quantitative assessment of the bioluminescence signal for different groups during the follow-up (*** indicates p=0.0002 post third treatment) (c) Assessment of decrease in the bioluminescence signal of the treatment group after each treatment cycle and (d) Kaplan-Meier survival plot of the various groups (**p=0.0067).
[00029] Figure 15 illustrates different complexes of oxasmaragdyrins along with possible structural/functional modifications.
[00030] Figure 16 illustrates chemical structures known in the art which exhibit NIR fluorescence and which can be employed in Photothermal therapy.
[00031] It should be appreciated by those skilled in the art that any diagrams herein represent conceptual views of illustrative systems embodying the principles of the present invention.

DETAILED DESCRIPTION

[00032] For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates. It will be understood by those skilled in the art that the foregoing general description and the following detailed description are explanatory of the invention and are not intended to be restrictive thereof.
[00033] Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” or “in an exemplary embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
[00034] The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method.
[00035] The present invention discloses BF2-Oxasmaragdyrin based Nanoparticles. These nanoparticles are similar to quantum dots as they possess photoluminiscent properties. However, they are not categorically identical to quantam dots as these nanoparticles do not possess semiconductor properties. With regard to present invention terms like ‘Tritolyl-BF2-Oxasmaragdyrin based Nanoparticles (TBSNPs)’ and ‘Tritolyl-BF2-Oxasmaragdyrin based Quantum dots (TBS-QDs)’ are used interchangeably.
[00036] The present invention relates to a process to synthesize meso-Tritolyl-BF2-Oxasmaragdyrin based quantum dots (TBS-QD). The precursor, Meso-Tritolyl-BF2-Oxasmaragdyrin, required to synthesize Meso-Tritolyl-BF2-Oxasmaragdyrin based quantum dots, is prepared using a reported method (Rajeswara Rao, M.; Ravikanth, M. Boron Complexes of Oxasmaragdyrin, a Core-Modified Expanded Porphyrin. J. Org. Chem. 2011, 76, 3582–3587). The reported method comprises of mixing 0.157 mmol of oxasmaragdyrin in 30 ml dichloromethane (CH2Cl2) to form a solution, subsequently adding triethylamine (6.28 mmol) at room temperature to the solution, followed by adding 7.85 mmol of Boron trifluoride diethyl etherate after 5 minutes and stirring the resultant mixture for 30 minutes. The reaction mixture is then diluted with CH2Cl2 and washed with 0.1 M NaOH and water. The organic layers are combined and dried over Na2SO4 followed by filtration of resultant mixture. The remaining solvent is evaporated on a rotary evaporator under vacuum and the resulting crude product is purified by column chromatography on alumina by using petroleum ether/dichloromethane (70:30). A green powder of meso-Tritolyl-BF2-Oxasmaragdyrin is obtained after purification. Further, smaragdyrin molecules with different complexes and functional groups (as illustrated in figure 15) can be synthesized and these precursors can be further used to prepare respective nanoparticles or quantum dots.
[00037] An embodiment of the present invention discloses a process to synthesize TBSQDs. The method comprises of synthesizing meso-Tritolyl-BF2-oxasmaragdyrin (TBS) using the reported method as disclosed earlier. Followed by preparing TBSQDs by thin-film hydration followed by high-pressure homogenization (HPH at 700-800 bar for at least 7-10 passes (Figure 1). The obtained TBSQDs suspension is characterized by Raman spectroscopy, TEM, and absorption and fluorescence spectroscopy.
[00038] Another embodiment of the present invention discloses a process to synthesize meso-Tritolyl-BF2-Oxasmaragdyrin based quantum dots (TBS-QD). The TBS-QD are prepared by subjecting the TBS under pressure using high pressure homogeniser in presence of a lipid (e.g. Phospholipids and Sphingolipids). The lipids may also be selected from Soya S100, S90, S85, S80, Soy Lecithins, DSPE, Phosphatidylcholines/ Phosphatidylethanolamines/ Phosphatidylinositols and PEGylated lipids. The formed TBS-QDs absorb in visible Near infrared region (Vis-NIR) and emit in Near infrared region (NIR) region. The synthesized TBS-QDs are photostable in NIR region. TBS-QDs are biocompatible and TBS-QDs are used for NIR imaging and therapeutic agents.
[00039] In yet another embodiment of the present invention Meso-Tritolyl-BF2-Oxasmaragdyrin based quantum dots (TBS-QDs) are prepared by synthesizing Meso-Tritolyl-BF2-Oxasmaragdyrin (TBS) precursor, followed by preparing a mixture of TBS (1 mg) and Lipoid S-75 (150 mg) in an organic solvent (20 mL) selected from chloroform, dichloromethane, ethanol, acetone, methanol, chloroform, USP Class 2/3 solvents. A thin film of said mixture is formed in a round bottom flask by evaporating the organic solvent on rotary evaporator for at least 30 minutes. The film is further hydrated with water (50 mL) atleast at 50°C for at least 1 hour on the rotary evaporator. A dispersion is formed by homogenizing the hydrated film at high pressure (850 bar) using a high-pressure homogenizer for at least 7-10 passes to obtain 0.1mg/mL solution of TBS-QDs in water and the obtained TBS-QD solution is characterized by Raman Spectroscopy, Transmission Electron Microscope (TEM), and Absorption and Fluorescence spectroscopy.
[00040] The TBSQDs sample was excited on the surface of silver colloidal particles with a 532 nm laser to minimize the effect of fluorescence on weak Raman signals. Sharper Raman signals were observed from the TBSQDs compared to that of the TBS (Figure 2a) with about 40-60% decrease (Table 1) in full width half maximum (FWHM) values from TBS to TBSQDs supporting the notion that the material was quantum confined. Further, TEM analysis of the sample demonstrated that the TBSQDs were amorphous/polycrystalline in nature with a particle size of 7.1 ± 2.1 nm (Figure 2b-d, Figure 3) which is less than the calculated Bohr radius (a0 ˜ 15 nm) for the material. The formation of quantum dots is attributed to the p-stacking of TBS under high pressure and stabilization of the hydrophobic self-assembly in water via the lipid matrix. The lipid disperses the TBS in the aqueous system and acts as a stabilizer during processing and storage. Elimination of the lipid from the reaction failed to disperse the TBS in water, and no TBSQDs were observed.
[00041] Photophysical properties: Absorption and emission studies were carried out on the synthesized TBSQDs. The absorption spectrum of the TBSQDs have maxima at the same wavelengths as TBS, but the bands are significantly broadened. Characteristic Soret band and four Q-bands are observed (Figure 4a), but with molar extinction coefficients that are 10 times lower than TBS (Figure 5, Table 2). Spectral broadening and decrease in absorbance at maxima are attributed to aggregation of the TBS within the nanoparticles. Further, a bathochromic shift of 30 nm in the last Q-band absorption maximum was observed for TBSQDs compared to TBS suggesting strong interaction between the TBS molecules. The emission spectra of the TBSQDs revealed that the TBSQDs fluoresce at 708 nm with a quantum yield of 0.09 and an excited state lifetime of 5.8ns. Interestingly, the fluorescence emission wavelength of TBSQDs was hypsochromically shifted by 4 nm, compared to that of the TBS (Figure 4b) though a bathochromic shift of 30 nm was observed in the absorption spectrum (Figure 4a). This indicates that the emissive species in the nanoparticles may be quite similar to TBS itself, even though aggregation has definitely happened, as indicated by the significant red shift in absorption spectra. This contention draws support from a comparison of fluorescence excitation spectra of TBS and TBSQDs (Figure 6 and 7). The excitation spectrum of the TBSQDs was practically indistinguishable from the normalized absorption and excitation spectra of TBS. The red-shifted band in the absorption spectrum was not observed. No fluorescence was observed when TBSQD was excited at 730 nm. Consequently, the absorption band at 730 nm is assigned to non-fluorescent aggregates of TBS. Interestingly, the fluorescence lifetime of TBSQDs was greater than that of TBS (Figure 4c, Table 2), indicating that the fluorescent species experience a more hydrophobic environment than TBS in water. On the other hand, fluorescence quantum yield decreased from 0.15 in TBSto0.09 in TBSQDs (Table 2). Hence, it may be inferred that the number of fluorescent species decreases upon aggregation, but the molecules that are fluorescent, possess longer lifetimes and fluorescence quantum yield. The net decrease in quantum yield is a result of the interplay of these two factors.To further probe into the excited state dynamics of the TBS dye and TBSQDs, ultrafast femtosecond transient absorption studies were performed. Pump laser of 400 nm, with energy of 400 nJ per pulse, was used to excite the samples, and the resultant data is presented in Figure 4d-m along with the fitting parameters in Table 3. The recovery of ground state bleach reflects the repopulation of the ground state after excitation with laser via nonradiative and radiative channels. Here, from the fitting parameters of the TBS dye at 440 nm and 469 nm, it is evident that the recovery occurs via two main pathways. A faster nonradiative recovery channel with a lifetime of 2.5 ps (28%) and 4.5 ps (37%) was observed at probe wavelengths of 440 and 469 nm, respectively. A longer component of >2 ns with 72% and 63% contribution is observed respectively at 440 and 469 nm. This long component is attributed to a S1-S0 radiative recovery channel, which was also observed in the TCSPC experiments (Figure 4c) with a fluorescence lifetime of 4.5 ns. Similarly, the decay curve of TBSQDs shows an additional faster nonradiative recovery channel of 0.7ps (40 %) and 0.5 ps (35%) along with 16.3 ps (35%) and 8.5 ps (32%), respectively, at 440 nm and 469 nm. The longer lifetime component of >2ns with 25% and 18% contribution is observed, respectively, when probed at 440 and 469 nm, which was also observed in TCSPC experiments with a fluorescence lifetime of 5.8 ns. In comparison, the total contribution from nonradiative recovery channels has increased from 28% to 75% from TBS to TBSQDs at 440nm and 37% to 82% at 469 nm. This increase in nonradiative recovery channels is attributed to the aggregates of TBS. Since they account for up to 75-82% of ground state recovery, the aggregates appear to bear promise in generating excellent photo-thermal response from TBSQDs when irradiated with the laser source (750 nm). TBSQDs possessed high stability post irradiation with a pump laser of 400 nm, whereas the TBS was photobleached within 3 scans. The latter resulted in poor signal to noise ratio at 710 nm for TBS dye, which restricted further probing the dynamics. The higher photostability of TBSQDs, facilitated longer irradiation exposure (15 scans of 1 hour each) and consequently provided improved S/N ratio at 712 nm and 724 nm. TBSQDs when probed at 712 nm, the kinetics followed similar trend as of 440 nm and 469 nm with one radiative decay channel with lifetime of >2 ns (22%) and two nonradiative decay channels with 1.6 ps (45%) and 48.3 ps (33%). Whereas two nonradiative recovery channels were observed when probed beyond 720 nm. The absence of a long lifetime component beyond 720 nm suggests that there is no fluorescence beyond 720 nm, which also corroborates with the absence of fluorescence when excited at 730 nm in steady state experiments
[00042] Photothermal studies
a. Photo-thermal transduction potential: The increased amount of nonradiative decay channels from TBS to TBSQDs led to the exhibition of excellent photo-thermal properties , and is determined by TBSQDs concentration, laser power, and time of irradiation. The central composite design (CCD), employed to assess the interaction between factors, along with the experimental results aretabulated (Table 4). The quadratic model was selected (p=0.0215), and the ANOVA indicated the selected model significant (p <0.0001), and the lack of fit insignificant (p <0.2797) (Table 5). The diagnostic tests did not reveal any anomalies, and the impact of various factors on response is provided in Equation (1),
Y = +58.33 + 8.69A + 8.23B + 7.36C + 1.67AC + 4.45BC – 3.72B2 (1)
where A, B, C, and Y represents concentration of TBSQDs, laser exposure time, laser power, and temperature, respectively. The synergistic and antagonistic effects of factors are indicated by positive and negative signs, respectively. Coefficients of single, two factors, and second-order represent the effect of individual, interaction, and quadratic nature, respectively. Equation 1 clearly indicates the concentration of TBSQDs has the highest impact on photo-thermal transduction potential, followed by laser exposure time and laser power.
3D plot was constructed to assess the impact of various factors (Figure 8a), by freezing laser irradiation time to 5 min. The numerical and graphical optimizations were carried out to attain the temperature of 52-65 °C, and independent variables ranges were set accordingly. The design space was evaluated by randomly performing experiments in the region, and it was observed that the results were well within the predicted ranges, as shown in Table 6.
b. Photo-thermal conversion efficiency (PCE): The PCE of material indicates the efficacy of material in converting the absorbed light to heat and is primarily influenced by the material absorption properties. The time vs. temperature profiling of TBSQDs and Milli-Q is given in Figures 8c and 8e, respectively.The PCE of TBSQDs was evaluated in accordance with the reported procedure, and the PCE of TBSQDs was found to be 43.6%.
c. Photo-thermal stability: The stability of a photo-thermal agent upon exposure to a light source is an essential requirement, and organic dyes are more prone to photo-bleaching. To evaluate the stability of TBSQDs, the material was subjected to NIR laser exposure multiple times, and it was observed that the material hadretained its photo-thermal properties even after fourcycles of NIR laser exposure (5 min each), as shown in Figure 4f. To further evaluate the stability, the absorbance and fluorescence spectra were recorded, and no spectral differences were observed. These studies indicate that the material has excellent photo-thermal properties, conversion efficiency, and photostability. The photostability is of particular interest, as most of the organic dyes are prone to photo-bleaching. These properties make TBSQDs a promising theragnostic agent for cancer imaging andtherapy.
[00043] Invitro studies
a. Biocompatibility: Invitro biocompatibility of TBSQDs was assessed in a mouse fibroblast L929 cell line, by estimating the intensity of fluorescent resorufin. Viable cells convert non-fluorescent resazurin to red-colored resorufin, and the cell viability was then estimated by quantifying the fluorescence of resorufin. As shown in Figure 9a, viability was more than 80% at a concentration of 150 µg/mL. Furthermore, cells treated with TBSQDs did not exhibit any abnormal morphological change, indicating excellent biocompatibility of the material.
b. Hemocompatibility: Hemocompatibility of a material is an absolute requirement for any parenterally administered material and is often influenced by size, morphology, and surface charge of material. The varying concentration of TBSQDs incubated with RBCs showed <5% hemolysis (Figure 9b), which is in accordance with the American Society for Testing and Materials (ASTM) E2524 guidance. The positive control (triton X-100) ruptured the red blood cells (RBCs) completely, and no pellet was observed during centrifugation (Figure 9c). Furthermore, ESEM analysis revealed no abnormalities, and biconcave morphology was preserved in TBSQDs treated samples indicating hemocompatibility of the material (Figure 9d-f).
c. Cellular Uptake: Fluorescence of the TBSQDs was utilized to quantify the uptake of TBSQDs by the cells at different time points in 4T1 breast cancer cells. Flow cytometric analysis was performed to sort cell populations with similar physical and/or chemical properties. Thus, facilitates the analysis of cells that have taken up the designed fluorescent material. Cells incubated with TBSQDs for different time periods indicated that uptake was immediate (i.e., 1-6h), reached a maximum at 12h, and was near saturated at the 24h time point. This was evident from the shift of the median fluorescence towards right in comparison to the negative control with increasing incubation time (Figure 10a). These results indicate that the designed formulation can be efficiently taken up by the cells, and 12h incubation is sufficient for efficient internalization of the QDs to perform in vitro photo-thermal efficacy testing of the material. The cellular uptake was also evaluated with confocal laser scanning microscopy (CLSM). The TBSQDs were internalized in the cells (Figure 11) and exhibited strong fluorescence in the NIR region (680-740 nm). DAPI staining was performed to assess localisation of the TBSQDs in the cells. The TBSQDs were found to be predominantly localises in the cytoplasm where they displayed excellent emission in the NIR region.
d. In vitro photo-thermal efficacy: To advance a material to preclinical assessment, in vitro efficacy must be evaluated. The photo-thermal potential of the TBSQDs was evaluated in 4T1 cells utilizing two different concentrations of the material (7.5 and 10 µg/mL) and varied laser exposure duration (3, 5, and 7 min). As shown in Figure 10b, 7.5 µg/mL of the material exposed to 3 min laser irradiation did not induce appreciable cell death (<5%)with 650mW laser power but, showed considerable cell death (>40 and 80%) at a longer laser exposure time of 5 and 7 min respectively. However, cells incubated with 10 µg/mL of the material and exposed to 650 mW laser power even for 3 min showed significant (p=0.019) cell death >70%. The cell viability further reduced to 15 and 5% (p = 0.0002), by increasing the laser exposure time further to 5 and 7 min respectively. The laser and material controls had viability greater than 95%. This clearly highlights that the designed formulation had potential for advancing it to preclinical testing in a mice tumor model.
e. Annexin V-PI Staining: Annexin V-PI staining is the gold standard technique to identify the mechanism of cell death post cytotoxic trigger, wherein single (Annexin V) and dual stained (Annexin V-PI) populations represent early and late apoptotic populations respectively, indicating cleaner mechanism of cell death whereas PI stained population refers to necrosis. Our study with the TBSQDs formulation indicated that the PTT treatment group showed >90% increase in the dual positive population, suggesting that the majority of the cells are in the late apoptotic stage (Figure 10c). However, only cells, cells+TBSQDs, and cells+laser control groups showed minute positivity for Annexin V, PI, and dual stain (Annexin V-PI) accounting for basal cell death.
[00044] Invivo studies
a. NIRF whole-body imaging and in vivo tumor homing: Whole-body imaging using NIR probes is exceptionally advantageous as tissue autofluorescence in this window is minimized. As the present formulation showed good fluorescence after cellular uptake in CLSM studies, it was tested in as a whole-body imaging agent. Live animal fluorescence imaging was done post intravenous (IV) administration of the TBSQDs. Post 24h of administration, a very strong fluorescence (p=0.0001) could be seen throughout the body of the animal {(4.2×107) ? 10 fold higher than the background body signal (3.0×106)}, indicating that such material could be efficiently utilized in combination with different targeting moieties for targeted fluorescence imaging approaches (Figure 12a-c). Simultaneously, tumor homing ability of the designed formulation was also evaluated. Animals used for whole-body imaging were also investigated to evaluatethe enhanced permeability and retention (EPR) based tumor homing potential of TBSQDs. Time-dependent fluorescence imaging revealed that initially, after material administration (2-24h), the fluorescence was observed throughout the body of the mice. However, at later times points 48-72h, a portion of the material accumulated in the tumor, taking advantage of leaky tumor vasculature (Figure 12d). To further quantify the amount of accumulated material, the tumor tissue was harvested, lysed, and extracted with dimethylsulfoxide (DMSO). The fluorescence of extracts revealed a significant {(p=0.013) 1-1.5µg/g} accumulation of the material in the tumor tissue (Figure 12e). Although partial EPR based passive tumor homing can be seen in our studies, the quantity deposited would not be sufficient for the effective tumor eradication via IV administration of the material followed by PTT. Hence, intratumoral material administration was utilized in subsequrnt in vivo PTT experiments. Nevertheless, the utilization of such materials as targeted photo-thermal and imaging agents in the future would be intriguing.
b. Biodistribution and single dose toxicity evaluation: Understanding the biodistribution and associated toxicity, if any, of the designed nanoformulation in preclinical rodent models is imperative, in the absence of the human data. Hence, biodistribution of TBSQDs was performed in Swiss mice post IV administration of the formulation. Ex vivo fluorescence imaging of the vital organs (lung, liver, spleen, kidney, and heart) on 14th day post material administration reveal that a major fraction of the material was deposited in these organs (Figure 13a-b). Qualitative fluorescence imaging and absolute quantification of organ accumulated material was performed by extracting the compound from the lysates of these organs. The data revealed that maximum accumulation was in the liver and spleen. Additionally, lower accumulation was seen in lung, kidney, and heart suggesting rigorous uptake of the material by reticuloendothelial system (RES) components accounting for inefficient tumor homing (Figure 13c-d). A significant portion of the material was not recovered, possibly due to body clearance of the formulation while in circulation. The acute toxicity of the material was also assessed by H&E staining of the vital organs and serum biochemical analysis for markers of normal liver and kidney function. The histopathological analysis showed no overt changes in the organs of the material injected and control animals (Figure 13e-f). Additionally, post 72h of material administration serum levels of alanine transaminase, aspartate aminotransferase, creatinine and urea in the treated mice were not perturbed and was similar to that of the control animals. These results indicate that the injected formulation has no adverse effect on the normal functioning of vital organs (Figure 13g). Collectively, these studies revealed that the formulation is biocompatible and non-toxic nature potentially is well suited for future clinical application in cancer therapy and diagnosis.
c. Preclinical photo-thermal efficacy study of TBSQDs: Photothermal therapy efficacy studies of TBSQDs in the preclinical settings was performed in the 4T1 breast tumor xenograft model. Tumor growth and reduction was monitored in a non-invasive manner using live animal bioluminescence imaging. TBSQDs administration was done on-site (intratumoral) in consideration of future intratumoral injection use inclinical applications of PTT. Efficacy studies with TBSQDs revealed that the formulation had excellent photo-thermal tumor ablation efficacy. The treatment regimen was done in 3 cycles; one treatment cycle (100µg nanoparticle and 6 min laser exposure at 650 mW) every second day for three days. Tumor ablation was observed after the first and second treatment cycle, resulting in reduction of the bioluminescence signal. Residual tumor at the treatment site, however necessitated another treatment. The third cycle of PTT treatment rendered complete tumor eradication with significant (p=0.0002) reduction in the bioluminescence signal (similar to the background signal). No accompanaying signs and symptoms of weight loss were observed suggesting no therapeutic burden (Figure 14). Control and material control group animals showed progressive tumor growth to day 5, following which the tumors in these groups started becoming necrotic, and the bioluminescence signal exceeded the detection capacity of the instrument. Hence, bioluminescence imaging was discontinued for these groups, but the animals were followed for survival studies and were sacrificed within day 10 due to ethical requirements. Animals belonging to the treatment group were monitored to day 30, wherein none of the animals in this group showed any signs of tumor relapse, thus demonstrating a significant (p = 0.0067) increase in the disease-free longevity for the PTT treated mice. Hence, the in vivo study conclusively illustrated the compatibility and the efficacy of the formulation for PTT in the preclinical application and provides a strong base forfuture clinical application.
[00045] TBSQDs were prepared by a simple experimental procedure at room temperature and thoroughly characterized the material by SERS, TEM, absorption, fluorescence and transient absorption spectroscopic studies. TBSQDs exhibited NIR fluorescence with an increased amount of nonradiative decay compared to TBS leading to excellent photo-thermal properties. The photo-thermal properties of the TBSQDs was accompanied by exceptional thermal stability and resistance to photobleaching. Furthrmore, in vitro studies revealed excellent biocompatibility, hemocompatibility, excellent cellular internalization, and photo-thermal efficacy. The subsequent in vivo studies demonstrated the non-toxic nature of the material post IV administration, and emanating fluorescence was exploited for NIRF-based whole-body imaging. The strong photo-thermal characteristics of TBSQDs were effectively utilized for tumor growth inhibition in the 4T1 xenograft mice model. The organic-based image guided photo-thermal therapy herein described provides as alternative approach to theragnostic agents and may find future use in clinical assessment of hyperthermia and whole-body imaging.
[00046] Although the present disclosure has been described in the context of certain aspects and embodiments, it will be understood by those skilled in the art that the present disclosure extends beyond the specific embodiments to alternative embodiments and/or uses of the disclosure and obvious implementations.

,CLAIMS:We Claim:
1. A process for synthesizing quantum dots, the process comprising:
preparing a mixture of meso-Tritolyl-BF2-Oxasmaragdyrin and a lipid in an organic solvent;
forming a thin film of the mixture by removing the organic solvent under pressure;
hydrating the film at an elevated temperature for a specific time;
homogenizing the hydrated film at a high pressure to obtain a dispersion; and
subjecting the dispersion to high shear homogenization followed by high pressure homogenization to obtain an aqueous solution of Meso-Tritolyl-BF2-Oxasmaragdyrin based quantum dots (TBS-QDs).
2. The process as claimed in claim 1, wherein the lipid is selected from Lipoid S-75, S100, S90, S85, S80, SoyLecithin, Phosphatidylcholines, Phosphatidylethanolamines, Phosphatidylinositols, PEGylated lipids or a combination thereof.
3. The process as claimed in claim 1, wherein the organic solvent is selected from chloroform, dichloromethane, ethanol, acetone, methanol, chloroform, USP Class 2/3 solvents.
4. The process as claimed in claim 1, wherein the temperature is at least 50°C.
5. The process as claimed in claim 1, wherein the high pressure homogenization is carried out atleast at 800 bar.
6. The process as claimed in claim 1, wherein the high pressure homogenization is carried out at least for 7-10 passes.
7. The process as claimed in claim 1, wherein the TBS-QDs absorb in visible Near infrared region(Vis-NIR) and emit in Near infrared region (NIR) region.
8. The process as claimed in claim 1, wherein the time is in the range of 30 minutes to 1 hour
9. The process as claimed in claim 1, wherein the hydrated film is homogenized at a high pressure of at least 700 bar.
10. The process as claimed in claim 1, wherein the particles size of TBS-QD is less than 10 nm.