The instant application claims the benefit of US Provisional Patent Application serial number 63/275,682, filed November 4, 2021, entitled “Fabrication of Smart Tantalum Carbide MXene Quantum Dots with Intrinsic Immunomodulatory Properties for Treatment of Allograft Vasculopathy”, the entire contents of which are incorporated herein by reference for all purposes.

BACKGROUND OF THE INVENTION

Low-dimensional carbon-based nanomaterials are unquestionably the “wonder materials” of today. Since the discovery of graphene in 2004, graphene and its derivatives have been studied extensively in electronic circuits, energy storage, light processing, chemical processing, and biomedical applications. ^{1 1 fi|} More recently, zero-dimensional graphene and MXene quantum dots have been found to possess broad immunomodulatory activity through interactions with a variety of immunologically active cells. ^{17 121} In particular, newer MXene- based quantum dots (MQDs) have potential to offer improved dispersibility, tunability, and biocompatibility over traditional graphene materials while maintaining immunomodulatory bioactivity. ^{1 12 151} However, the field remains in a relative infancy and the detailed mechanisms of action of these materials have remained elusive so far. ^[16] Furthermore, currently available evidence is largely based on in vitro studies and MXene materials have not yet been explored in vivo in a clinically relevant inflammatory disease model.

Recently, we reported the biocompatibility and anti-inflammatory effects of titanium carbide (TisC2T _x ) MQDs in low concentrations. ^[11] Notably, these MQDs effectively suppressed pro-inflammatory THI polarization of naive CD4 ⁺ T-lymphocytes under synthetic in vitro conditions. These revelations have sparked significant interest in immunoengineering TFCbTx MXenes to tackle today’s clinical challenges. ^[16] In particular, MXene-based approaches are being developed to treat refractory inflammatory conditions and suppress rejection of transplanted tissue constructs. ^{[11 16]} However, the long-term bio-inertness of titanium-based materials has since been called into question. ^{117 181} In fact, several reports on the cytotoxicity of ThCbTx MXene at medium-to-high concentrations raised significant concern on the eventual clinical translatability of these materials. ^[19 ’ ^20] Future application of this technology therefore hinges on addressing this fundamental limitation.

In response to this challenge, other MXene compositions, such as niobium carbide (NbzC), have been developed with reduced cytotoxic potential. ^{121 231} However, niobium has not been commonly used in biomedical applications and their long-term safety remains poorly understood. ^[24] On the other hand, tantalum-based biomaterials are well studied and have been previously shown to possess improved corrosion resistance, biocompatibility, and bioactivity over those derived from titanium. ^{125 271} In particular, tantalum oxides have been shown to be more stable and inert than their titanium-based counterparts, which contributes to the excellent biological safety profile of tantalum-based materials. ^{128 341} These finding have been corroborated by both in vitro and in vivo experiments showing the safety of high dose tantalum carbide (Ta4CsT _x ) MXene nanosheets. ^[35,36] However, the successful synthesis of highly desirable Ta4C?T _x MQDs has not been reported yet. MQDs are uniquely suitable for biomedical and immunoengineering applications due to their improved aqueous stability and subcellular-level interactions. ^[37] Development of Ta4C?T _x MQDs is therefore urgently needed to keep pace with this rapidly evolving field.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a method for synthesis of MXene-based quantum dots comprising: etching a quantity of a tantalum carbide MAX phase powder, thereby providing MXene nano sheets; sonicating and homogenizing the MXene nanosheets, thereby providing multilayer, oligolayer and monolayer flakes; and hydro thermally treating the multilayer, oligolayer and monolayer flakes, thereby providing MXene-based quantum dots.

In some embodiments of the invention, the MXene is tantalum carbide.

According to another aspect of the invention, there are provided tantalum carbide MXene-based quantum dots comprising surface hydroxyl, carboxyl, chlorine, fluorine, and amine based functional groups.

According to another aspect of the invention, there are provided MXene-based quantum dots prepared according to the method described above.

According to another aspect of the invention, there is provided use of the tantalum carbide MXene-based quantum dots described above for reducing immune cell infiltration. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Synthesis schematic model, stoichiometry, and materials characterization.

(A) Step-by-step schematic on the conversation of Ta4AlCs MAX phase bulk to zerodimensional Ta4C?T _x MQDs using a facile protocol. (B) Proposed reaction chemistry for the synthesis of Ta4C3T _x MQDs, including surface modification with tantalum oxides (TaO2 and Ta2O5). (C, D) Morphology and microstructural characterization of the Ta4CsT _x MNSs and MQDs. (C, left and middle) High-resolution TEM (HRTEM) images of multilayer Ta4C?T _x nanosheets revealed well-defined and exfoliated crystals with lattice d-spacing of ~ 0.260 nm. (C, right) Furthermore, the corresponding SAED pattern of the nanosheets depicted a uniform hexagonal crystallization pattern typical for MXene materials. (D, left) HRTEM images of Ta4C?T _x MQDs displayed proper synthesis of particles with high surface functionalization after hydrothermal treatment at 180 °C. (D, middle) As shown, the average diameter of a single T A/iTx particle is ~ 3.5 nm, which is ideal for targeted subcellular applications. (D, right) The lattice d-spacing of MQDs was found to be ~ 0.338 nm. Collectively, these data support the successful design and production of the new Ta4CsT _x MQDs.

Figure 2: Characterization of the structure, functional groups, and chemical composition of as-synthesized Ta Tx MQDs. (A-C) XRD phase characterization of Ta4C?Tx MQDs was performed at 5° to 80° 20. Our XRD data displayed the main characteristic (002) peak of the MXene materials at ~ 7° 29 in the Ta4C3T _x MQDs. High-resolution XRD analysis of the MQDs depicted a remarkable downshift of the Al-containing peaks, which was a robust confirmation for the synthesis of Ta4CsT _x MXenes. The peaks were mainly matched according to the standard reference codes for Ta carbide, Ta-oxide carbide and Ta oxides phases (96- 210-3218, ICSD156383, and a-alumina). (D) FTIR analysis of the MQDs. Our FTIR data showed clear formation of additional functional groups on the surface of MQDs. The FTIR spectra displayed a broad peak at ~ 3500 cm ^-1 , which may be attributable to expansion of lattice parameters in the MQDs. (E) The wide-scan XPS spectrum of these MQDs revealed that the main Al 2p and Al 2s peaks at the binding energies of 64 to 80 eV and 115 to 125 eV were significantly extracted from the material composition during synthesis process. Our XPS survey also showed well-defined MXene characteristics (Ta 4f, Ta 4p, C Is, O Is, Cl 2p, Na Is, and F Is) with a high degree of surface functionalization in comparison with its MAX phase structure. (F to H) Furthermore, the narrow spectra of Ta 4f, C Is and O ls confirmed that Ta4C?T _x MQDs were successfully synthesized. Overall, the high-resolution narrow spectra of TaX^Tx MQDs are supported by our XRD and other characterizations.

Figure 3: Thermophysical and optical absorption properties ofTa s x MQDs. (A to C) TGA/DSC analysis of Ta^Tx MQDs in argon and atmospheric conditions. (A) Under argon gas, TGA analysis of Ta4CsT _x MQDs demonstrated no significant effects on the surface terminations or decomposition of material after annealing of Ta^Tx MQDs at up to 800 °C. (B) Furthermore, our data showed that there was no significant mass loss after 350 °C, and the char residue was higher than 90% in this temperature range. (C) However, under normal atmospheric conditions, the TGA curve of Ta^Tx MQDs showed a steady increase in mass percentage due to the oxidation process after ~ 600 °C. (D) The UV-Visible spectrum of the Ta^CsTx MQDs demonstrated a strong dose-dependent absorption in the area of ~ 300 nm, corresponding to the lateral carbon structure, and an additional broad absorption peak at around 900 nm. (E and F) The calculated a for the novel Ta4CsT _x MQDs was measured to be 0.525 L-g ’em ¹ and 0.573 L-g ’em ¹ at 404 and 808 nm, respectively. (G) The zeta potential of Ta4CsT _x MQDs was largely negative across the pH range of 2 to 12 (around 0 to -30 mV). A slight increase in surface charge of the MQDs was noted at pH higher than 10, likely due to property changes in the functional groups of particles under strong alkaline conditions.

Figure 4: Evaluation of the Reactive Oxygen Species (ROS) Generation and Biocompatibility of Ta-tCsTx MQDs. (A) Total cellular ROS was evaluated in HUVECs using a green fluorescence probe. (B) Culture with T A/iTx MQDs at concentrations of 2 to 100 pg- mL ¹ did not increase total cellular ROS when compared to the control. (C) Additionally, caspase-3/7 activation was assessed using a green fluorescent probe, and Ta4C?T _x MQDs at concentrations of 2 to 100 pg-mL ¹ did not increase apoptotic activation when compared to the control. (D) Biocompatibility at 3 days was assessed at different MQD concentrations (0.5 to 20 pg-mL ¹ ) using the LDH release assay. No significant increases were observed in maximum LDH release between MQD-treated groups and the control. (E) Biocompatibility at 7 days was assessed at different MQD concentrations (0.5 to 20 pg- m ¹ ) using the WST-1 Cell Proliferation assay. No significant differences were observed in cellular proliferation between the MQD-treated groups and the control.

Figure 5: In vitro Evaluation of Immunomodulatory Effects ofTa-iC lx MQDs. (A) Schematic representation of the in vitro immunomodulatory model. Ta4C ₃ T _x MQDs interact with HUVECs to reduce inflammatory activation of co-cultured lymphocytes. (B) Cytotoxicity assessment of Ta4C ₃ T _x MQDs on human lymphocytes. No significant cytotoxicity was observed when lymphocytes were treated with 2 pg-mE ¹ of MQDs. (C) Schematic showing the timeline for in vitro immunomodulation assessment. HUVECs were pre-treated with Ta^CsTx MQDs, activated with IFN-y, and then co-cultured with human lymphocytes for 9 days. (D) Flow cytometric gating strategy for analysis of immunomodulation. Single lymphocytes were gated on the CD3 ⁺ CD4 ⁺ gate. (E) Intracellular staining for IFN-y and IE-4 were used to identify THI and TH2 T-helper cells respectively. (F) Treatment with 2 pg-mE ¹ of Ta^CsTx MQDs reduced the percentage of THI cells when human lymphocytes were co- cultured with activated HUVECs. (G) No significant differences were observed in the percentages of TH2 cells in the co-culture experiment.

Figure 6: Mechanistic Evaluation of the Immunomodulatory Effects of l'a^s l x MQDs. (A) Eight microscopy demonstrated that Ta ETx MQDs were readily internalized into HUVECs after 24 hours of culture. (B, C) Quantitative PCR analysis was used against genes involved in antigen presentation, cellular adhesion, lymphocyte recruitment, and chemokine signaling. Activation of HUVECs using IFN-y resulted in an increase in pro-inflammatory signaling. No significant differences were observed between cells treated with 20 pg- m ¹ of Ta^CsTx MQDs and the vehicle control. (D) Treatment with Ta4C ₃ T _x MQDs was found to alter the expression of the T-cell co-inhibitor PD-L1 and the T-cell co-activator CD86 on the surface of activated HUVECs. A significant increase was noted in the endothelial expression of PD-L1, and a trend towards a decrease of CD86 was observed after treatment with 20 pg- mL ¹ of Ta ₄ C ₃ T _x MQDs. (E) Schematic representation of the immunomodulatory mechanisms of Ta ₄ C ₃ T _x MQDs. MQDs are internalized into cells through active endocytosis, after which their surface architecture facilitates endosomal escape. They then participate in immunomodulatory signaling to alter the ratio of surface co-activator and co-inhibitors, which subsequently results in reduced T-cell activation.

Figure 7: In vivo Evaluation of the Immunomodulatory Effects of Ta-iC lx MQDs using a Rat Aortic Allograft Vasculopathy Model. (A) Schematic representation of the model. The descending thoracic aorta was transplanted from male Lewis rats into the abdomens of male Sprague-Dawley rats. Animals received a tail vein injection of 1 mg- kg ¹ body weight of Ta^CLTx MQDs and were kept for seven days. (B) Intraoperative photograph showing the transplanted aortic segment. Arrows represent the proximal (black) and distal (white) anastomoses. (C, D) H&E staining of explanted abdominal aortic segments. Obvious signs of inflammation could be observed in the transplanted groups. Furthermore, there appeared to be quantitative reductions in the degree of endothelial thickening and adventitial lymphocyte infiltration in the MQD-treated group when compared to the vehicle control (insets and arrows).

Figure 8: Quantitative Assessment of the in vivo Immunomodulatory Effects of Ta Tx MQDs. (A) Immunohistochemistry against a-SMA showed significant disruption in the media of the transplanted aortic segments amongst transplanted animals, which was ameliorated with treatment using Ta^Tx MQDs. (B) a-SMA was quantified using mean fluorescence intensity and normalized against a segment of the non-transplanted thoracic aorta from each animal. The vehicle control demonstrated a significant drop in the amount of a- SMA, which was ameliorated with treatment using Ta4C?T _x MQDs. (C) Quantitative reductions were observed in the infiltration of CD8 ⁺ T-lymphocytes (shown in red) in the adventitia of transplanted aortic segments between the Ta4CLT _x MQD-treated group when compared to the vehicle control group. (D, E) Flow cytometric analysis of the circulating T- lymphocytes of animals. Single lymphocytes were gated on the CD3 ⁺ CD4 ⁺ gate. As shown here, there appeared to be lower numbers of CD25 ⁺ regulatory T-lymphocytes in the aortic transplant group, which was ameliorated with treatment using 1 mg- kg ¹ body weight of intravenous Ta4C?T _x MQDs.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

MXene nanomaterials have sparked significant interest amongst interdisciplinary researchers to tackle today’s medical challenges. In particular, colloidal MXene quantum dots (MQDs) offer the high specific surface area and compositional flexibility of MXene while providing improvements to aqueous stability and material-cell interactions. The current study reports for the first time the development and application of immunoengineered tantalumcarbide (Ta^CsTx) MQDs for in vivo treatment of transplant vasculopathy. This report comes at a critical juncture in the field as poor long-term safety of other MXene compositions challenged the eventual clinical translatability of these materials. Using rational design and synthesis strategies, our Ta4C?T _x MQDs leverage the intrinsic anti-inflammatory and anti- apoptotic properties of tantalum to provide a novel nanoplatform for biomedical engineering. In particular, these MQDs are synthesized with high efficiency and purity using a facile hydrofluoric acid-free protocol and are enriched with different bioactive functional groups and stable surface TaCh and Ta20s. Furthermore, MQDs are spontaneously taken up into antigen- presenting endothelial cells and alter surface receptor expression to reduce their activation of allogeneic T-lymphocytes. Finally, when applied in vivo, Ta^CsTx MQDs ameliorated the cellular and structural changes of early allograft vasculopathy. These findings highlight the robust potential of tailored Ta^Tx MQDs for future applications in medicine.

Herein, we present the design, fabrication, characterization, and application of immunoengineered tantalum carbide (Ta4C3T _x ) MXene quantum dots for in vitro and in vivo immunomodulatory applications. These Ta^Tx MQDs were rationally designed for biomedical applications through a tailored etching, exfoliation, and hydrothermal process. As- synthesized MQDs exhibited high concentrations of MXene surface functional groups as well as the surface tantalum oxides (TaCh and Ta2Os), which contributed to its excellent biocompatibility with human cells. In particular, high concentrations of Ta^Tx MQDs did not induce oxidative stress and cytotoxicity in cultured human endothelial cells (ECs). Furthermore, these MQDs were spontaneously internalized into ECs and mechanistically contributed to reducing the immunogenicity of these cells through regulation of T-cell activation. Finally, when applied in an in vivo model of organ transplant rejection, intravenous administration of Ta4C?T _x MQDs reduced both immune cell infiltration and structural degeneration within transplanted tissues. Taken together, this study highlights the future potential of tailored Ta4CsT _x MQDs in immunoengineering and other biomedical applications. Rationale, Design, and Synthesis of Ta^ sTx MQDs

The engineering of biomedically relevant nanomaterials requires strict control of their chemical composition, structure and properties. ^[13] The deliberate choice of tantalum-based MXene in this study arises from considerations of both biocompatibility and bioactivity. Despite ample evidence on the biomedical efficacy of TFCLTx MXene nanosheets and quantum dots, increasing concerns on their potential cytotoxicity, albeit at higher doses, cannot be ignored. Large quantities of exposed titanium oxides (TiCh and Ti2Ch) can form on the surface of TisC2T _x MQDs during the hydrothermal process or upon aqueous dispersion. ^{138 401} Additionally, TFCbTx MXene products can spontaneously oxidize under ambient storage conditions to form transition metal oxide particles. ^[41] The presence of titanium oxides is particularly concerning in designing materials for biomedical applications, as it can catalyze the production of reactive oxygen species (ROS) and generate oxidative stress to nearby cells and tissues. ^{142 441} This surge in ROS also induces the release of pro-inflammatory cytokines from resident tissue macrophages, which hinders the functionality of immunomodulatory materials.

Therefore, in the current study, Ta4CsT _x MQDs were specifically designed with biomedical applications in mind and synthesized using a facile methodology to accommodate these design requirements. The Ta4AICL MAX Phase was chemically etched and exfoliated to form accordion-like Ta4CsT _x MXene nanosheets (MNSs) using hydrochloric acid/sodium fluoride (HCl/NaF) as etchant. The resultant MXene products were subsequently dispersed in pure distilled water and further treated by bath sonication to obtain multi-, oligo- and singlelayered Ta4CLTx MXene nanocrystals. Finally, the obtained aqueous colloidal suspension underwent hydrothermal treatment at 180 °C for 12 hours to obtain zero-dimensional Ta4CsT _x MQDs. A step-by-step schematic of the production of Ta4CsT _x MQDs is presented in Figure 1A. Despite milder nature of the HCl/NaF etchant over hydrofluoric acid (HF), effectiveness of this etching process has already been demonstrated for niobium and vanadium carbide (V2C) MXenes, which carry similar formation energies to Ta4C?T _x MXene. ^{145 471} Furthermore, there are several distinct advantages to this approach. First, the fluoride salt etchant is expected to produce fewer surface defects than HF treatment, thereby reducing opportunities for oxidative degradation and increasing the stability and shelf-life of the end product. ^[41,48] Second, this etching process facilitates intercalation of cations and water between the MXene layers, thereby weakening interlayer interactions. ^[48] This results in expansion of the interlayers spacing in MXene nanosheets and facilitates the subsequent delamination process. Lastly, this approach reduces the manufacturing challenges associated with use of concentrated HF while maintaining the strict tunability of the MXene end products. In this study, NaF was specifically chosen over the conventional LiF due to cytotoxic concerns associated with lithium moieties in the structure of MXenes. ^[44]

Furthermore, the Ta4C?T _x MXene nanosheets were treated by ultrasonication and subsequent homogenization to enhance its specific surface area and aqueous colloidal dispersibility. In particular, mechanical vibration and/or sonication treatment increases the degree of cationic intercalation and further increases interlayer spacing. As a result, the obtained colloidal solutions contained well dispersed and electrostatically stabilized MXene nanosheets. Furthermore, colloidal suspensions of MXene flakes produced from this process are less likely to clump or aggregate, thereby increasing its accessibility for further functionalization. ^[49] This protocol, therefore, offers potential for the industrial development of bioactive and clinically translatable Ta4CsT _x MQDs.

According to another aspect of the invention, there is provided a method for synthesis of MXene-based quantum dots comprising: etching a quantity of a tantalum carbide MAX phase powder, thereby providing MXene nanosheets; sonicating and homogenizing the MXene nanosheets, thereby providing multilayer, oligolayer and monolayer flakes; and hydro thermally treating the multilayer, oligolayer and monolayer flakes, thereby providing MXene-based quantum dots. The MXene may be tantalum carbide.

The tantalum carbide may be Ta4C3T _x .

In some embodiments, the MAX phase powder is etched with HC1 and NaF.

In some embodiments of the invention, the MAX phase powder is etched with 6-12 M HC1 and 2-5 M NaF at about 40-65 °C for about 24-72 hours.

In some embodiments of the invention, the hydrothermal treatment comprises heating to about 120-180 °C for about 6-24 hours.

According to another aspect of the invention, there is provided carbide MXene-based quantum dots comprising surface hydroxyl, carboxyl, chlorine, fluorine, and amine based functional groups.

In some embodiments of the invention, the tantalum carbide MXene-based quantum dots further comprise stable surface tantalum oxides (Ta20s and TaCh) and alkali metal component (Na).

In some embodiments, the tantalum carbide MXene-based quantum dots have an average Ta4C?T _x diameter of about 3.5 nm.

In some embodiments of the invention, the tantalum carbide MXene-based quantum dots have lattice d-spacing of about 0.338 nm.

In some embodiments of the invention, the tantalum carbide MXene-based quantum dots have a surface charge between -5 to -10 mV at pH 7.

According to another aspect of the invention, there are provided MXene-based quantum dots prepared according to the method described above.

The MXene may be tantalum carbide.

The tantalum carbide may be Ta4C3T _x .

According to another aspect of the invention, there is provided use of the tantalum carbide MXene-based quantum dots as described above for reducing immune cell infiltration.

Furthermore, as discussed herein, in our body, endothelial cells (ECs) act as the barrier between blood and tissues. ECs play a critical role in the rejection of transplanted organ. After transplantation of donor organ/cells, recipient ECs are activated and alert recipient immune system, leading to immune activation, vascular injury (transplant vasculopathy), and subsequent rejection of the allograft (donor organ). Recruitment of inflammatory immune cells is critical to development and progression of allograft rejection. Surprisingly, we found that tantalum quantum dots significantly reduced the percentage of inflammatory immune cells after co-culture with activated ECs.

We also found that because of the high abundance of negatively charged hydroxyl, carboxyl, chlorine, fluorine, and amine based functional groups on the surface of Ta quantum dots, these were rapidly taken up by ECs. This ultimately allows them to interact with different components/proteins within the ECs and promote immunomodulatory signaling.

The invention will now be further elucidated and/or explained by way of examples; however, the invention is not necessarily limited to or by the examples.

Example 1 - Proposed Reaction Chemistry for Synthesis of Ta CsTx MQDs

The proposed chemical reactions for the synthesis of Ta4C?T _x MQDs in the abovedescribed fabrication process are presented in Figure IB. Exfoliation of the Ta4AIC? MAX phase was achieved using a two-step approach. First, the MAX phase powder was chlorinated by treatment with 12M HC1 at 60 °C to significantly remove the surface Al layer through formation of aluminium chloride (AlCh). Additionally, the presence of NaF in the etching solution completed the exfoliation process through the formation of sodium hexafluoro aluminate (NasAlFe), further removing any remaining Al traces. This led to successful production of multilayered Ta4CsT _x MXene nanosheets (Figure IB, Equation 1). Moreover, the presence NaF during the agitation process resulted in further surface functionalization of the MXene layers with abundant -OH groups. Furthermore, the proposed reaction chemistry also supports effective fluorination of the end product with rich -F surface terminals. Together, these mechanisms of reactions enabled facile exfoliation of Ta AlCs MAX phase powder and efficient synthesis of two-dimensional Ta CsTx MXene nanosheets (Figure IB, Equation 1 to 3).

The chemical reactions that occurred during functionalization process of the colloidal dispersions arose after partial oxidation of Ta^Tx MNSs in aqueous media (Figure IB, Equation 4). The hydrothermal process led to formation of tantalum oxide (both TaCh and Ta20s) layers on the surface of Ta4CsT _x MQDs, presumably through a secondary crystal nucleation mechanism (Figure IB, Equation 5 and 6). It is important to note that these chemical reactions also facilitate the formation of additional -OH and =0 groups on the surface of MQDs after hydrothermal treatment. Additionally, chemical interactions of existing C1-, F-, and Na-based compounds with the surface of Ta4C?T _x are expected to occur during the synthesis process as well. Therefore, different stable surface functional groups can be readily identified on the surface of MQDs, supporting its chemical stability and bioactivity.

Example 2 - Microstructural Characterization of Ta CsTx MQDs

Successful synthesis of Ta^Tx MQDs using our innovative synthetic process was confirmed using transmission electron microscopy (TEM), fast Fourier transform (FFT) analysis, selected area diffraction analysis (SAED), and energy-dispersive X-ray spectroscopy (EDS). SEM images of the Tau Cs MAX phase and its corresponding EDS analysis showed bulky morphology with an atomic percentage of 19.77 % for Al in the composition. TEM images of Ta^CsTx nanosheets after treatment with HCl/NaF demonstrated stacked basal planes of the MXene layers (Figure 1C, left). Additionally, high resolution TEM (HRTEM) and SAED images of exfoliated Ta^Tx MXene flakes confirmed a symmetric crystalline structure consisting of layers with d-spacing of ~ 0.260 nm, which can be assigned to (111) plane (Figure 1C, middle and right). Notably, the obtained hexagonal lattice d-spacing of Ta4CsT _x MXene nanosheets agreed well with previous reports, confirming that the planar structure of sheets remained stable and unperturbed during the synthesis process. ^[50] Furthermore, EDS elemental and mapping analysis of this sample clearly depicted significant removal of Al from the structure of MAX phase.

The subsequent mechanical processing and hydrothermal treatment at 180 °C resulted in successful formation of surface functionalized Ta^Tx MQDs (Figure ID, left). HRTEM images of the acquired MQDs revealed well-defined quantum structure with a particle size of less than 5 nm in diameter (Figure ID, middle). Furthermore, Ta^Tx MQDs exhibited a highly crystalline diffraction pattern with multiple differently oriented planes. In particular, the corresponding SAED/FFT patterns displayed a crystalline structure of MQDs with an atomic d-spacing of ~ 0.338 nm (inner plane: ~ 0.238 nm, Figure ID, right). Notably, amorphous rings were seen in the SAED analysis of Ta^Tx MQDs and can be attributed to remaining non-crystalline carbon-based particles. ^[38] Finally, EDS elemental analysis of Ta^Tx MQDs retained the low atomic percentage of Al at less than 1%, which is consistent with what was seen in the MNSs. Together, these observations provide robust evidence that the HF-free etching and subsequent hydrothermal treatment employed in this study has successfully fabricated crystalline Ta4C?T _x MQDs.

To further characterize the structural transformation of MAX phase to Ta4C?T _x MQDs, X-ray diffraction (XRD) analysis was performed. Our data demonstrated that the main characteristic (002) peak of MXene clearly emerged at ~ 7° 20 in the Ta4C?T _x MQDs sample (Figure 2A and B). Simultaneously, the Ta4AlCs peaks were significantly downshifted after the exfoliation and hydrothermal process. In particular, one of the main MAX phase peaks at ~ 16° 29 was completely removed from the XRD spectra of Ta4C3T _x MQDs. Additionally, a minor amorphous curve between 10° to 30° 29 was identified in the XRD pattern of Ta4C?T _x MQDs (Figure 2B and C). As described in the previous section, this change reflects the remaining carbon dots formed during the hydrothermal process. Furthermore, a contamination peak of tantalum carbide (Ta2C) in the XRD spectrum of the MAX phase at ~ 50° 29 disappeared completely in the XRD spectrum of Ta4C?T _x MQDs, reflecting the efficiency of synthesis and purity of the end product.

A new dominant peak was seen at -12° 29 in the Ta4C?T _x MQDs, corresponding to the addition of a crystalline tantalum carbide-based oxide composite in the structure of MQDs. Identification of alpha-alumina (01-AI2O3) peaks in the XRD pattern of MQDs provided further evidence for effective removal of Al and the conversion of its remaining traces to oxide form. Additionally, there were two new precise peaks which emerged in the XRD spectra of the MQDs, corresponding to TaCh (110) and Ta20s (010). The detection of transition metal oxide formation during hydrothermal process reflects both theoretical and experimental evidence available in the literature. ^[38] Furthermore, the XRD pattern of the Al-etched Ta4CsT _x revealed enlarged lattice spacing in the atomic structure of MQDs (Figure 2B). This expansion is largely attributed to surface functionalization during the synthesis process. Thus, these characterizations demonstrated successful production of surface modified Ta4C?T _x MQDs.

Next, the surface functional groups of Ta4C?T _x MQDs were evaluated using Fourier- transform infrared spectroscopy (FTIR). The FTIR spectrum of Ta4C?T _x MQDs identified characteristic Ta-C, Ta-O, and Ta-F bonds of Ta4C?T _x MXenes (Figure 2D). Additionally, FTIR assessment revealed the vibrations of key surface functional groups, including -OH, C- F, C=O, Ta-C, Ta-O, and Ta-F, available in the structure of Ta4C?T _x MQDs. The FTIR stretching of these bonds was detected at the wavelengths of ~ 500 nm to 3500 nm. The presence of a C-F peak in the spectrum of Ta4CsT _x MQDs at ~ 1200 cm ^-1 suggested efficient fluorination of particles during synthesis process. Additionally, a weak vibration was identified at ~ 3100 cm ^-1 that may suggest the presence of amine functional group (-NH2). ^[11] This characterization confirmed the presence of quantities of function groups on the surface of Ta^CsTx MQDs and indicated the successful fabrication of functionalized particles.

Lastly, X-ray photoelectron spectroscopy (XPS) was used to characterize the surfacebonding structures of Ta4CsT _x MQDs. Wide-scan survey comparison of Ta4AIC? MAX phase and its derived product revealed the generation of high quality MQDs during the synthesis process. (Figure 2E). In particular, the Al 2p narrow scan XPS spectrum of Ta4C?T _x MQDs confirmed the efficacy of etching process in removing Al layers from the structure of the MAX phase. Additionally, the high-resolution C is spectrum of TaALTx MQDs contained the combination of Ta-C _x , C-C, C-N, C=C, C-O, and C=O peaks fitted at binding energies between 280 and 290 eV (Figure 2F). Additionally, the O Is spectrum identified oxygencontaining peaks located between 526 eV to 536 eV, signifying a high level of oxygencontaining functional groups on the surface of these MQDs (Figure 2G).

The Ta 4f narrow scan of Ta4CsT _x MQDs displayed prominent 4f 5/2 and 4f 7/2 peaks of tantalum and tantalum oxide at the binding energies of 20 to 30 eV (Figure 2H). In particular, formation of lateral species of Ta ⁴⁺ and Ta ⁵⁺ as main Ta-oxide structures (TaCh and Ta2Os) was detected at binding energies of ~ 24 to 25 eV. Furthermore, in agreement with the proposed chemical reactions, XPS high-resolution spectra of Cl 2p, Na Is, and F Is demonstrated the formation of additional surface functions on MQDs by the HCl/NaF etchant. In particular, the Cl 2p spectrum exhibited a combination of metal-chloride peaks (3/2 and 1/2) at binding energies of ~ 197 to 200 eV and non-metal Cl at ~ 201 eV. Moreover, the XPS spectra of Na Is depicted a dominant peak at ~ 1071 eV assigned to Na ⁺ ions. This region contains two peaks at the binding energies of ~ 1067 and 1069 eV corresponding to the interaction of Na ⁺ with Ta-oxide and Ta-C, respectively. Additionally, as discussed in the previous section, the rationally designed synthesis protocol enabled a mechanism to fluorinate the surface of Ta4CsT _x MQDs, as confirmed by the high-resolution F Is spectrum. Of note, two peaks were fitted at binding energies of ~ 684 and 687 eV, attributed to Ta-F (atomic percentage: 71.82%) and C-F (atomic percentage: 28.18%) respectively. These measurements served as another confirmation to successful surface modification of Ta4C?T _x MQDs during the synthesis process. Overall, the XPS analysis of as-synthesized Ta^CsTx MQDs agreed very well with our XRD and FTIR characterizations and confirmed that the employed synthesis procedure reported in the current study was highly efficient and suitable for targeted applications.

Example 3 - Thermal, Optical and Surface Properties of Ta-iC lx MQDs

The surface properties of MXene nanostructures are defined by their synthesis conditions. In this regard, effective fabrication methods must be applied to obtain MXene materials with the desired surface terminations and long-term stability. Previously, thermogravimetric analysis (TGA) has been used to characterize the temperature-dependent desorption of surface terminations of MXene materials. ^[51] This method can effectively quantify the thermal stability of surface functional groups and terminations after annealing. In the current study the stability of Ta^CsTx MQDs was evaluated using TGA. Under vacuum condition, the -OH functional group is the first species to desorb from the surface of heated MXenes, starting at temperatures above 300 °C. ^[52] Notably, the signal of -OH desorption from the MXene samples can be significantly masked by the release of deprotonated H2O during TGA measurement. However, de-functionalization of surface -OH groups in the structure of MXenes subsequently lead to partial electron transformation of this group into more stable oxygen-containing functional species. Our TGA curve of Ta4CsT _x MQDs depicted a slight mass-loss between ~ 150 and 300 °C, and its char residue was 10% at around 600 °C (Figure 3A). However, our TGA data showed almost no mass loss after 350 °C, and in this temperature range, its char residue was as higher than 91% (Figure 3B).

Interestingly, our TGA data demonstrated that annealing Ta^CsTx MQDs up to 800 °C had no significant effects on its surface termination and did not result in significant decomposition of material. A minor mass gain (~ 1%) was observed at annealing temperature above 800 °C and can most likely be attributed to oxidation of impurities or slight decomposition of the material. In contrast, TGA curve of Ta4CsT _x MQDs generated under normal atmospheric conditions showed a continuous increase in its mass percentage due to oxidation process, starting at ~ 600 °C (Figure 3C). As apparent in our results, the heat flow data of differential scanning calorimetry (DSC) curves are in good agreement with the obtained TGA in both argon and atmospheric conditions (Figure 3B, C). Together, this data support that the employed HF-free protocol in the current study was able to successfully synthesize highly stable Ta4C?T _x MQDs.

Furthermore, the MXene materials are relatively transparent in visible lights and are known to possess excellent optical properties. ^[53] These specific properties precisely depend on the tailored intercalation, interlayer spacing, and surface architecture of individual MXene materials. Therefore, surface modification and functionalization during the synthesis of MXene structures can be used to achieve desired absorption and optical properties. To this regard, we assessed the UV-Visible absorption spectra of an aqueous colloidal dispersion of Ta^CsTx MQDs. In particular, MQDs at different concentrations of ~ 30 to 300 pg-mL ¹ were examined to characterize their optical properties. The UV-Visible spectrum of Ta4C?T _x MQDs at 230 nm to 990 nm demonstrated a clear dose-dependent absorption profile for the dispersed particles (Figure 3D to F). Strong absorption was noted in the area of ~ 300 nm, corresponding to the lateral carbon structure of Ta4C?T _x MQDs. In fact, due to the colloidal nature of Ta4C3T _x MQDs, a linear correlation between absorption and concentration of particles could be observed for the MQDs, which can be described using the Beer-Lambert law. Based on the measured standard curves, the a value was calculated to be 0.525 L-g ’em ¹ and 0.573 L-g ’em ¹ at 404 and 808 nm, respectively, which can be used as a robust parameter for future studies using Ta4C?T _x MQDs. Subsequently, the long-term colloidal stability of aqueous MQDs dispersion was further confirmed six months after the initial synthesis and characterization. As shown in these optical micrographs, the developed environment-friendly protocol in the current study resulted in the fabrication of stable surface-modified and uniform MQD suspensions without significant stacking and agglomeration of the particles at the test concentrations of 250 pg- mL ¹ .

The surface charge of synthetic nanomaterials also has a significant effect on their surface bioactive properties. ^[54] In the next experiment, the surface charge behavior of the Ta4CsT _x MQDs was assessed at a concertation of 75 pg-mL ¹ and different pH values. The zeta potential (Q data in the current study suggested that as-synthesized Ta4C?T _x MQDs have a surface charge between -5 to -10 mV at pH 7 (Figure 3G). However, the MQDs exhibit pH- dependent change in their surface charge, with the point of zero charge (PZC) at a pH of approximately 2 and progressively more negative surface charge at higher pHs. This observation can be explained by the abundance of surface carboxyl groups which demonstrate pH-dependent ionization, and is consistent with the previously reported analysis of other MXene counterparts. ^[55] Notably, the zeta measurements in the current study revealed a slight increase (around 10 %) in the surface charge of the MQDs from pH 10 to 12, which may be attributed to changes in the structure of particles or surface functional groups under strong alkaline conditions. Nevertheless, the negative surface charge of these Ta4C?T _x MQDs likely contributes to the bioactivity of these quantum dots through facilitation of material-cell interactions. Taking all these accounts together, the data strongly supports the successful development of a new nontoxic Ta /iTx MQDs functional material with excellent microstructure and surface properties for targeted biomedical and other potential applications, including theranostic, cancer therapy, regenerative nanomedicine, electronic, and water filtration.

Example 4 - Biocompatibility of Ta CsTx MQDs

The biocompatibility of as-synthesized Ta^Tx MQDs was assessed in vitro using cocultures with human umbilical vein endothelial cells (HUVECs). The endothelial cells (ECs) form the lining of blood vessels and serve as the first point of contact between the body and intravenously delivered nanomaterials. These cells also play important roles in the regulation of inflammation, coagulation, and nutrient delivery to different tissues. Endothelial toxicity can therefore significantly limit the future biomedical applications of nanomaterials. ^[23] Furthermore, previous studies have reported that other forms of MXenes, such as ThCbTx MXenes, can increase cellular ROS levels, create oxidative stress to nearby cells, and cause cellular damage. ^19,39 ’ ⁴¹ This increase in ROS also induces the release of pro- inflammatory cytokines from resident tissue macrophages and interferes with antiinflammatory and immunomodulatory properties of implanted biomaterials in the body. ^[56] Therefore, in the current study, we first assessed if the presence of Ta^Tx MQDs causes any ROS generation in cells. The HUVECs were cultured with or without different doses of Ta^CsTx MQDs (2 to 100 pg-mL ¹ in PBS) for 24 hours. The intracellular ROS levels were assessed using the CellROX green fluorescent dye. It is evident from our data that Ta^Tx MQDs did not increase intracellular ROS levels across the concentration range used in this study (Figure 4A and B). In fact, the highest concentration of MQDs (100 pg- mL ¹ ) appeared to attenuate the oxidative stress compared to vehicle control group (Figure 4A and B). These data highlight the unique advantages afforded by the structural composition of Ta-based MXenes over their titanium counterparts.

Next, the biocompatibility of Ta ^Tx MQDs with HUVECs was investigated using the CellEvent fluorescence-based apoptosis detection kit that detects the activities of caspase-3 and caspase-7. These two caspases are the primary executioners of programmed cell death in cells subject to insurmountable stressful conditions. The activation of both caspase-3 and caspase-7 are reported to be associated with cellular apoptosis and cell death. In the current study, HUVECs were subjected to nutrient deprivation and subsequently cultured with Ta^CsTx MQDs for 24 hours. As shown in Figure 4C, Ta4C ₃ T _x MQDs up to 100 pg-mL ¹ exhibited no significant activation of caspase-3 and caspase-7 when compared with the controls. Therefore, Ta ₄ C ₃ T _x MQDs are cyto-compatible and do not cause any cellular damage. Furthermore, amongst the concentrations used for subsequent studies (0.5 to 20 pg-mL ¹ ), no significant differences were observed in cellular cytotoxicity and proliferation at any TaX^Tx MQD concentrations for up to 7 days (Figure 4D, E). These findings further establish the importance of rationally designed and synthesized Ta^C^Tx MQDs for future biomedical applications.

Example 5 - Immunomo dulatory Properties of Ta4C T _x MQDs

The immunomodulatory properties of Ta4C ₃ T _x MQDs were investigated in vitro using co-cultures of activated HUVECs and human peripheral blood mononuclear cells (PBMNCs). As the barrier between blood and tissues, ECs play a critical role in the pathophysiology of organ transplant rejection. After allo-transplantation (donor-derived), ECs are activated and act as antigen-presenting cells to the recipient immune system, leading to immune activation, vascular injury, and subsequent rejection of the allograft (donor organ). ^[57,58] In particular, recruitment of pro-inflammatory type 1 T helper (THI) cells is critical to development and progression of allograft rejection. ^[59] Thus, in this study, we examined the immunomodulatory effects of Ta ₄ C ₃ T _x MQDs using activated HUVECs, PBMNCs, and THI cells as a model for organ transplant rejection (Figure 5A). First, Ta4CsT _x MQDs at a concentration of 2 pg- mL ¹ were cultured with antibody- activated and Tnl-directed human PBMNCs in the absence of HUVECs to look for direct immunomodulatory effects. We have previously reported for the first time that titanium carbide (TisC2T _x ) MQDs display direct immunomodulatory effects. ^[11] Ta4C?T _x MQDs do not appear to exert statistically significant immunomodulatory effects on their own, in the absence of ECs as antigen-presenting cells, after 1 week of culture. Specifically, no differences were observed in the percentage of interferon-gamma (IFN-y) expressing T-lymphocytes (Control 74.3%, MQDs 76.3%, ns) or in the proliferation of T-lymphocytes (Control 36.8-fold, MQDs 37.3-fold, ns). Furthermore, these experiments also confirmed the biocompatibility of Ta CLTx MQDs with human lymphocytes, with no obvious differences in T-cell viability seen after 1- week of culture (Control 83.4%, MQDs 83.2%, ns; Figure 5B). These findings are congruent with previous reports on graphene quantum dots (GQDs), which also possess no direct immunomodulatory effects on lymphocytes and require antigen-presenting cells to exert their effects. ¹⁸¹

To test this hypothesis, HUVECs were treated with Ta4CLT _x MQDs at 2 pg-mL ¹ for 24 hours prior to activation with IFN-y at 10 units- mL ¹ for 24 hours. Robust activation was achieved at this time point with significant upregulation of human leukocyte antigen class II (HLA-DRa). These cells were subsequently co-cultured with PBMNCs in medium containing Ta4C?T _x MQDs at 2 pg-mL ¹ and interleukin-2 (IL-2) at 5 ng- mL ¹ for 9 days (Figure 5C). As shown in Figure 5D to 5G, Ta4C?T _x MQDs exerted distinct immunomodulatory effects on T- lymphocytes through activated HUVECs. In particular Ta4C?T _x MQDs significantly reduced the percentage of IFN-y ⁺ THI cells amongst the CD4 ⁺ T-lymphocyte population after coculture with activated HUVECs (Vehicle 17.7%, MQDs 14.9%, p<0.05; Figure 5F). These effects were not seen in the unactivated HUVEC group (Vehicle 12.15%, MQDs 12.04%, ns). Interestingly, no significant differences were observed in the proportion of interleukin-4 (IL-4) expressing type 2 T helper (TH2) cells amongst co-cultures with both activated (Vehicle 1.6%, MQDs 1.4%, ns; Figure 5G) and unactivated (Vehicle 1.9%, MQDs 1.7%, ns) HUVECs. These findings are in line with previous reports, which showed that GQDs interact with antigen-presenting dendritic cells to reduce the proportion of pro-inflammatory IFN-y ⁺ THI cells after in vitro stimulation. ^[8] However, unlike GQDs, Ta4C?T _x MQDs do not induce upregulation of TH2 T-lymphocytes. TH2 cells are known to exacerbate allergic reactions and contribute towards activation of the humoral immune system. ^160,611 Thus, these findings strongly support the hypothesis that Ta4CsT _x MQDs can produce beneficial immunomodulatory effects in clinically-relevant models.

Example 6 - Mechanism of Immunomodulation by Ta4C T _x MQDs

To understand the mechanisms of immunomodulation through Ta^Tx MQDs, the direct interaction of Ta4C?T _x MQDs with HUVECs were investigated. Interestingly, it was discovered in the current study that Ta4C?T _x MQDs were rapidly taken up by endothelial cells and localize near the nucleus of the cell (Figure 6A). While not wishing to be limited to a particular theory or hypothesis, the inventors believe that the abundance of negatively charged hydroxyl, carboxyl, chlorine, fluorine, and amine based functional groups on the surface of Ta4C?T _x MQDs might have facilitated this internalization. ^[62] Furthermore, pH dependent changes in the surface charge of Ta4C3T _x MQDs (Figure 3G) might have facilitated their endosomal escape shortly after internalization. ^[63] As Ta4C3T _x MQDs become less negatively charged, they can interact with the membrane of the endosomes to escape into the cytoplasm. This ultimately allows them to interact with nuclear and cytoplasmic proteins and participate in subsequent immunomodulatory signaling.

To gain insight into the mechanisms of immunomodulatory signaling induced by Ta4C3T _x MQDs, a quantitative polymerase chain reaction (qPCR)-based gene expression analysis of common immunologic pathways was performed in HUVECs (Figure 6B, C). As shown here (Figure 6B, C), Ta4C3T _x MQDs do not significantly alter expression of genes related to antigen presentation (IRF1, TAPI, HLA-A, B2M, HLA-DRa, CIITA), cellular adhesion (PECAM-1, VE-Cadherin), lymphocyte recruitment (VCAM-1, ICAM-1, E-Selectin, and P-Selectin), or chemokine signaling (CCL-2, CXCL9, CXCL10). Rather, a significant shift in the expression pattern of surface co-stimulatory and co-inhibitory molecules in ECs was observed in the current study. As shown in Figure 6D, there was a 3.3-fold increase in the expression level of the programmed death ligand 1 (PD-L1) in activated HUVECs treated with Ta4C3T _x MQDs compared with those treated with the vehicle (p<0.05). Simultaneously, there was a trend towards a 1.3-fold decrease in the expression level of the CD86 in activated HUVECs treated with Ta4C3T _x MQDs (p=0.18). Both PD-L1 and CD86 are reported to be involved in T-cell activation pathways via antigen presenting cells. PD-L1 acts as a coinhibitor to T-cell activation while CD86 acts as a co-activator. ^[64 ’ ^65] Therefore, by altering the relative expression of PD-L1 and CD86 in antigen-presenting endothelial cells, Ta^CiTx MQDs have the mechanistic potential to reduce host inflammatory activation against allogeneic organs and tissues (Figure 6E). These Ta4C?T _x MQDs are therefore promising materials for future applications in preventing allograft rejection and regenerative medicine. Example 7 - Application of Ta CsTx for In Vivo Immunomodulation

Finally, a rat model of allograft vasculopathy was used to explore the immunomodulatory effects of the synthesised TarCTTx MQDs in vivo. After solid organ transplantation, donor endothelial injury and activation results in the activation of alloreactive T-lymphocytes in the recipient. One of the pathologic mechanisms for ultimate loss of the allograft is the development of allograft vasculopathy. ^{166 681} This inflammatory condition uniquely manifests as accelerated narrowing of the blood vessels within transplanted hearts, lungs and kidneys. ^{169 71 1} Currently established treatments are largely ineffective and Ta^Tx MXene-based immunomodulation may offer promise as a novel therapy for this therapeutic challenge.

In the current study the descending thoracic aorta was harvested from male Lewis rats and transplanted as an interposition graft into the abdominal aorta of male Sprague-Dawley rats (Figure 7A, B). Ta4C?T _x MQDs at a dose of 1 mg-kg ^_1 bodyweight (or an equivalent volume of saline, for control animals) were injected through the tail vein immediately after the transplantation. Animals were followed for one week after surgery, during which no adverse effects were observed with respect to the physical appearance, behaviour, and body weight of animals. Blood and tissues were then collected for subsequent analysis. No gross histologic differences were noted in the lungs, liver and kidneys between the treatment groups. However, as shown in Figure 7C, histologic sections of the abdominal aorta from transplanted animals showed obvious inflammatory changes when compared with sham animals. Furthermore, significant differences were noted in both endothelial proliferation and adventitial immune cell infiltration between control and MQDs treated animals (Figure 7D, arrows). Animals treated with intravenous Ta4CsT _x MQDs appeared to have reduced endothelial injury and immune cell infiltration when compared with those injected with saline. To quantify the degree of vascular injury, immunohistochemistry was performed against alpha-smooth muscle actin (a-SMA), which is a marker for blood vessel integrity. An early sign of allograft vasculopathy is immunologic-mediated loss of a-SMA expressing medial smooth muscle cells. ^[72,73] Here, we noted a significant decrease in the amount of medial a-SMA within the transplanted aortic segments of control animals (Figure 8A). Furthermore, this loss of medial a-SMA appeared to be ameliorated in animals treated with intravenous Ta4C?T _x MQDs. When normalized against a segment of native thoracic aorta, transplanted aortic segments of treated animals displayed significantly better relative a-SMA expression than control animals (Vehicle 0.8-fold, MQDs 1.4-fold, p<0.0001; Figure 8B). Congruent with these observed changes, a higher number of infiltrating adventitial cytotoxic CD8 ⁺ T-lymphocytes were observed within transplanted aortic segments of control animals when compared with those treated with Ta^Tx MQDs (Figure 8C). Additionally, these findings were corroborated by flow cytometric identification of circulating CD4 ⁺ CD25 ⁺ regulatory T-lymphocytes (T _reg s) performed one week after transplantation (Figure 8D, E). The Tregs are known to play a significant role in the development of immunologic tolerance after transplantation and higher numbers of T _re gs is associated with reduced allograft vasculopathy after transplantation. ^[74] In our study, transplanted animals had a numeric drop in the number of circulating T _re gs (Sham 19.1%, Vehicle 15.5%, p=0.12) when compared with sham animals, which was ameliorated by treatment with Ta^Tx MQDs (Vehicle 15.5%, MQDs 20.9%, p<0.05). This supports the proposed hypothesis that treatment with Ta^Tx MQDs reduces immune activation, promotes allograft tolerance, and prevents immune- mediated damage of transplanted allogeneic vascular segments. Taken together, these findings are highly suggestive of an in vivo immunomodulatory role for Ta^Tx MQDs in the treatment of allograft vasculopathy.

In conclusion, the analysis within the current study presented the rational design, development and application of immunoengineered tantalum carbide (Ta^Tx) MXene quantum dots. As synthesized Ta^Tx MQDs exhibited high concentrations of functional surface groups to facilitate their role in biomedical applications. Upon in vitro testing, these Ta^CsTx MQDs exhibited a high level of direct interaction with human endothelial cells while maintaining excellent biocompatibility. In particular, Ta^CsTx MQDs are rapidly taken up into ECs and reduce their ability to activate allogeneic T-lymphocytes through regulation of surface co-activator and co-inhibitor molecules. Additionally, when applied in an in vivo model of allograft vasculopathy, Ta4C?T _x displayed strong immunomodulatory functions and reduced early development of allograft vasculopathy. This study for the first time highlights the strength and future potential of a rationally designed Ta4C?T _x MQDs in immunoengineering and other biomedical applications.

Example 8 - Experimental Section

Synthesis of Tc C lx MQDs

The zero-dimensional Ta4C?T _x MQDs were synthesized from Ta4AIC? MAX phase through etching, exfoliation, and subsequent hydrothermal process. First, bulky MAX phase was etched to synthesize 2D Ta4C?T _x MXene nanosheets using HC1 (216147, Fisher Scientific Co.) and NaF (>99%, Sigma Aldrich). Briefly, Ta4AIC? powder (Eaizhou Kai Kai Ceramic Material Co. Etd.) was slowly immersed and stirred in a mixture solution containing 12M HC1 and 4M NaF at 60 °C for 48 hours. The precipitated flakes were collected by high-speed centrifugation, followed by several washing steps with pure distilled water at 10,000 rpm for 15 minutes each. The collected precipitates were freeze-dried for 48 hours and then dried in an air oven at 60 °C for 24 hours. The resultant MXene nanosheets were further treated by bath sonication and probe homogenizer for 60 minutes and 15 minutes, respectively, to obtain multi-, oligo-, and monolayer flakes, before being further treated by the hydrothermal process at 180 °C for 12 hours. The collected aqueous MQDs suspensions were then sterilized using a steam autoclave and used for further experiments.

Physicochemical Characterization of TaA Tx MQDs

Morphology and microstructural properties of materials were characterized using FESEM (SEM 450, Thermo Fisher Scientific), TEM (FEI Talos F200X S/TEM, Thermo Fisher Scientific), EDS, FTIR (Nicolet Nexus 870, Thermo Fisher Scientific), XPS (PHI Quantera, Physical Electronics, Inc.), and XRD (Bruker diffractometer). X-ray diffraction peaks were collected in the range from 5 to 80° 20 using a continuous scan with a rate of 3°-mm ¹ and a report interval of 0.05°.

Thermogravimetric and Optical Analysis The TGA/DSC assessment of the Ta^CsTx MQDs was performed using a Q-600 SDT (TA-Instruments) on a DSC-TGA Standard Module at a heating rate of 10 ⁰ C-min ^-1 in air and argon (100 mL min). The temperature ramps up to 100 °C with a heating rate of 10 ⁰ C-min ^-1 , kept isothermal for 10 minutes, and ramps as high as 1000 °C. Furthermore, the optical properties of the aqueous Ta4C?T _x MQDs suspensions at a concentration of ~ 50 pg-mL ¹ were assessed by the Cytation5 Imaging Multi-Mode Reader (BioTek) at different excitationemission wavelengths.

Zeta Potential Measurements

The surface charge of an aqueous Ta4C?T _x MQDs colloidal suspension at a concentration of ~75 pg- mL ¹ was assessed using the Nanobrook ZetaPALS (Brookhaven Instruments) at different pH of 2, 4, 6, 8, 10, and 12. The pH of the aqueous MQDs was titrated with the addition of adequate amounts of 12M HC1 and 12M sodium hydroxide (NaOH) solutions. The electrical conductivity of aqueous Ta4C?T _x MQDs was adjusted at same concentration using 0.1 phosphate-buffered saline. The experiments were replicated for ten cycles, and the average values are reported.

Animals and Ethics

All animal protocols were approved by the University of Manitoba Animal Care Committee and conform to standards and guidelines set out by the Canadian Council on Animal Care. Male Lewis rats (260 to 280 grams) were used as donors and obtained from Charles River Laboratories. Male Sprague-Dawley rats (260 to 280 grams) were used as recipients and obtained from Central Animal Care Services at the University of Manitoba. All surgical procedures were performed at the R.O. Burrell Laboratory at the St. Boniface Hospital Research Centre, University of Manitoba, Winnipeg according to standard operating procedures.

Endothelial Cell Culture

Pooled human umbilical vein endothelial cells were obtained from Lonza (C2519A) and cultured in EGM-2 (CC-3162, Lonza) using manufacturer protocols unless otherwise specified. Cells were further characterized to express typical endothelial markers. Cells used for experiments were between passages 3 and 5 for all experiments.

Reactive Oxygen Species Assay Total cellular reactive oxygen species (ROS) were assessed using the CellROX Green Reagent (C 10444, Thermo Fisher Scientific). Briefly, HUVECs were plated on 96 well plates and grown to 80% confluency. They were then treated subject to a nutrient starvation for 24 hours by diluting the culture medium 1:1 with PBS in the presence of varying concentrations MQDs. Cells were then imaged on a Nikon Ti-2E fluorescence microscope and mean cellular fluorescence was quantified for 10 cells per high-powered field using ImageJ software. Three replicates were included for each treatment condition.

Caspase 3/7 Activity Assay

Caspase 3/7 activity was assessed using the CellEvent™ Caspase-3/7 Green Detection Reagent (C 10423, Thermo Fisher Scientific). Similar to the ROS assay, HUVECs were plated on 96 well plates and grown to 80% confluency. The cells were then subjected to a nutrient starvation for 24 hours by diluting the culture medium 1:1 with PBS in the presence of varying concentrations MQDs. Subsequently, the cells were treated with Hoechst 33342 (R37605, Thermo Fisher Scientific) to define the nucleus and imaged on a Nikon Ti-2E fluorescence microscope. Relative caspase activation was estimated based on the degree of nuclear fluorescence, with 10 nuclei quantified for each high-powered field using ImageJ software. Three replicates were included for each treatment condition.

Biocompatibility Assessment

Biocompatibility of the MQDs with HUVECs at 3 and 7 days was assessed using the Lactate Dehydrogenase (LDH) Cytotoxicity Detection Kit (MK401, Takara Bio). Briefly, HUVECs were plated on 96 well plates and grown to 80% confluency. They were then treated with varying concentrations of MQDs and grown for 7 days in culture. At 3 and 7 days, media was taken from the wells for LDH assessment. Six replicates were included for each treatment condition. Additionally, cell proliferation at 7 days was assessed using the WST-1 Cell Proliferation Assay kit (K304, BioVision Incorporated). Briefly, HUVECs were plated on 96 well plates and grown to 80% confluency. The cells were then treated with varying concentrations of MQDs and grown for 7 days in culture and used for the WST-1 assay according to manufacturer protocols. Five replicates were included for each treatment condition.

Assessment of Cellular Uptake HUVECs were plated on chamber slides and cultured with MQDs at a concentration of 20 pg- ml/ ¹ for 24 hours. The cells were then fixed with 4% paraformaldehyde and mounted using ProLong™ Diamond Antifade Mountant with DAPI (P36962, Thermo Fisher Scientific). The fixed cells were imaged on a Nikon Ti-2E fluorescence microscope using the bright-field mode and the DAPI filter.

Western Blot Analysis

Western blot analysis was used to confirm the induction of major histocompatibility complex-II (MHC II) expression. Briefly, HUVECs were treated with or without 20 pg-mL ¹ of Ta4C?Tx MQDs for 24 hours prior to induction with 10 units- mL ¹ of IFN-y. Cells were then scraped and collected in RIPA buffer for protein isolation and western blotting using standard protocols.

Immunomodulation Assays

HUVECs were plated on 24 well plates at a density of 20,000 cells per well and allowed to attach for 24 hours. The cells were then treated with MQDs at 2 pg-mL ¹ for 24 hours. Subsequently, HUVECs were activated using IFN-y (570202, BioLegend) at a concentration of 10 units- mL ¹ for 24 hours. Cells were then washed in preparation for subsequent co-culture experiments. Human peripheral blood mononuclear cells (PBMNCs) were isolated from whole blood obtained from healthy volunteers using Lympholyte®-H Cell Separation Media (CL5015, Cedarlane Labs). Co-cultures were performed in EGM-2 supplemented with 5 ng-mL ¹ of interleukin-2 (589102, Biolegend) and varying concentrations of MQDs for 9 days. At this point, cells were pulsed with the Cell Stimulation Cocktail with protein transport inhibitor (00-4975-93, Thermo Fisher Scientific) for 6 hours, after which PBMNCs were collected for subsequent flow cytometric analysis.

In a separate experiment, the direct immunomodulatory effects of Ta4C3T _x MQDs were investigated in the absence of HUVECs. Briefly, naive CD4 ⁺ T-lymphocytes were isolated through negative magnetic activated cell sorting using the MojoSort Human CD4 Naive T Cell Isolation Kit (480041, BioLegend). Cells were cultured in 24 well plates at a density of 10 ⁵ cells per well and stimulated with 10 pg-mL ¹ of plate-bound anti-CD3 antibody (300313, BioLegend) and 2 pg-mL ¹ of soluble anti-CD28 antibody (302913, BioLegend) at the start of culture immediately after isolation. Cells were grown in Advanced RPMI 1640 medium (12633012, Gibco) supplemented with 10% FBS (12483020, Gibco) 2 mM GlutaMAX (35050061, Gibco), 1:100 Penicillin- Streptomycin (15140122, Gibco), 0.055 mM 2- mercaptoethanol (M3148, Sigma- Aldrich) and 20 units- mL ¹ recombinant human IL-2 (589102, BioLegend). For THI polarization, the medium was also supplemented with 10 ng-mL ¹ recombinant human IL-12 (573002, BioLegend). The cells were cultured for one week and analyzed using flow cytometry.

Flow Cytometry

Cells were analyzed on the CytoFLEX Flow Cytometer (Beckman Coulter) with the appropriate fluorescence-minus-one and isotype controls. Data analysis was performed using CytExpert Software version 2.3.1.22 (Beckman Coulter, Brea, California).

Quantitative PCR Analysis

Total cellular RNA was isolated using the Aurum™ Total RNA Mini Kit (7326820, Bio-Rad) and quantified using a NanoDrop™ Spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized using the High-Capacity cDNA Reverse Transcriptase Kit (4368814, Thermo Fisher Scientific) using the manufacturer recommended protocol. qPCR was performed using the CFX384 Touch Real-Time PCR Detection System (Bio-Rad) with the appropriate no template controls.

In Vivo Aortic Transplantation Model

Animal care and anesthesia was performed using standard operating procedures at the University of Manitoba. After induction of anesthesia, donor Lewis rats underwent a median sternotomy where the mediastinal structures were removed. Immediately afterwards, the thoracic aorta was mobilized and harvested, taking care to mark the proximal end and to ligate branch vessels to ensure subsequent hemostasis. The donor aorta was stored in ice-cold saline for subsequent transplantation. Recipient animals underwent induction of anesthesia and median laparotomy. The abdominal viscera were displaced to access the retroperitoneum. The infrarenal abdominal aorta was isolated using careful dissection and branch vessels inferior to the gonadal arteries were ligated. Next, clamps were placed on the abdominal aorta and a segment was resected. The previously harvested donor thoracic aortic segment was then anastomosed as an interposition graft using two end-to-end anastomosis with 8-0 Prolene sutures. Hemostasis was ensured using a combination of pressure and SurgiCel. Good pulses were appreciated in the distal segment of the aorta prior to closure. The abdominal viscera was then replaced into the abdomen and the abdomen was closed in a routine fashion. Animals were kept for one week, after which point the transplanted aortic segment as well as a segment of the thoracic aorta were harvested from each animal for subsequent analysis. Blood was also collected at the time of harvest for flow cytometry.

Immunohistochemistry

After collection, tissues were fixed in 10% buffered formalin (SF100, Fisher Scientific) at room temperature overnight. The tissues were then embedded in paraffin blocks and sectioned to 5 pm thick sections and mounted on glass slides. Hematoxylin and eosin staining was performed in a regressive fashion with Harris’ hematoxylin using standard protocols. For immunohistochemistry, sections were rehydrated using a series of ethanol steps and then incubated overnight with primary antibody at 4 °C. The tissue sections on slides were then washed and incubated with secondary antibody for 1 hour at room temperature. Finally, the sections were mounted with Prolong Diamond Antifade Mountant with DAPI and imaged using a Nikon Ti-2E fluorescence microscope.

Statistical Analysis

Comparisons between two variables were performed using an unpaired two-tailed Students’ t-test. Comparison between three or more groups were performed using a one-way analysis of variance (ANOVA) and Tukey’s honestly significant difference test. Comparisons involving two independent variables were compared using a two-way analysis of variance and the Bonferroni multiple comparisons test. An adjusted p-value less than 0.05 was considered to be significant. All statistical analysis was performed using Prism version 9.02 for Windows (GraphPad, San Diego, California).

While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention. REFERENCES

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