1. IntroductionChemotherapy, a primary treatment for early and late stages of cancers, is often overrun by biological and metabolic barriers limiting its therapeutic accumulation at the cancer site. It instead induces harmful collateral necrosis of healthy cells, multi drug resistance and recurrent tumor growth [1]. Poor tumor specificity or bioavailability can be mitigated via anticancer prodrug delivery circumnavigating tumor heterogeneity and collateral damage to benign cells [2]. Cancer prodrugs, with significantly lower cellular toxicity can be catalytically metabolized into an active form, in situ, enhancing on site bioavailability, therapeutic ability and reducing side effects [3]. Many prodrugs can be activated by the tumor microenvironment, like thiols, reactive oxygen species, and acidity or triggered by enzymes in the tumor microenvironment like β-glucuronidase [4], carboxypeptidase [5], or cytochrome P450 [6]. These factors, lack transformational specificity, precluding reliable application [3]. Instead, using transition metal catalysts like ruthenium, palladium, copper, or gold in biorthogonal in situ bond cleavage-based in vivo prodrug transformations show superior efficiency, specificity and reliability [7,8]. Palladium (Pd(0)) has shown excellent catalytic ability and biocompatibility for in vivo prodrug activation of proteins like phosphothreonine lyase by decaging propargylcarbamate-protected lysine residues [9]. Other examples entail in vitro bioorthogonal catalytic synthesis of anti-cancer agent 5-fluorouracil from biologically inert precursor [10] or the development and bioorthogonal activation of palladium-labile prodrugs of gemcitabine [11]. Catalytic stability and efficacy of nanomaterial anchored Pd(0) heterogeneous catalysts in simulated biological conditions has made bioorthogonal chemistry a mainstay in current chemical biology research, spatially targeted, prodrug-based, site-specific cancer therapies [10].In this study, we examine a heterogenous Pd-anchored Resorcinol-formaldehyde-hyperbranched PEI nanostructure (MR-Pd) for in vivo, in situ catalytic Suzuki-Miyaura cross-coupling of iodoprazole and boronic ester to the anti-cancer agent, PP121 and its concomitant efficacy in 2D and 3D encapsulated structures containing PC3 cancer cells [12]. The conversion uses a mesoporous, palladium anchored mesoporous polymeric catalyst, synthesized as per our previous work [13]. The hydrogel for cell encapsulation comprises of GelMA-Sodium alginate combination, into MR-Pd is dispersed alongwith the non-cytotoxic cancer pro-drug precursors. In this work we have showed the feasibility of killing malignant cells, in a 3D encapsulated model, via in situ production of anti-cancer agent using cross coupling, under biological conditions. This can pave the way for an injectable hydrogel as a vehicle for possible introduction of catalyst-precursor combination at the tumor site. While the hydrogel sets under in vivo conditions, the same conditions allow for cross coupling of the pro drug precursors at in vivo temperatures. The anti-cancer agent PP121, thus produced in situ can kill cancerous cells by apoptosis. 3. Results and DiscussionsHere, we anchor Pd(0) in a mesoporous polymeric resin network of hyperbranched polyethyleneimine (PEI) by a one-pot, template-free synthetic methodology, employed in our earlier published work [13]. Resorcinol and formaldehyde polymerize, react with PEI’s amine groups forming benzoxazine rings, complexing and anchoring the Pd atoms in a mesoporous polymer network. This is also depicted in Scheme 1 below.The N2 adsorption, SEM (Supplementary Figure S1) confirm high surface area and porosity with a uniform microporous morphology and an interconnected network. The existence of Pd(0) is corroborated by XPS spectra while EDX elemental analysis confirm the presence of nitrogen atoms, from PEI, in the catalyst structure (Figure S1). We abbreviate the catalyst structure as MR-Pd. In our earlier work [13], we obtained a uniform dispersion of Pd nanoparticles without a template or cytotoxic organic solvents, paving way for scale up, low process toxicity and a better choice for biological applications. We also established good catalytic efficiency in mild conditions with Suzuki-Miyaura cross-coupling [13] achieving upto 98.98 ± 1% and 62 ± 5% conversion in batch and continuous microreactors, respectively. The chemistry tailorability makes its biological application possible via structural manipulation and/or dispersion in a carrier hydrogel.For in situ, biological catalyst application for cancer treatment, the cytocompatibility of the MR-Pd catalyst structure was shown by live- dead fluorescence and Actin-DAPI staining assays recording negligible cytotoxicity (Supplementary Figure S2) [14]. This was corroborated by quantified cell viability and cellular expansion with the mesoporous catalyst comparable to growth media as control (Figure S2).This cytocompatible, MR-Pd catalyst was now tested for in situ drug synthesis in a standard monolayer culture, seeded with PC3 prostrate cancer cells for two days yielding a monolayer with 50% confluency. Live-dead cell viability assay (Figure 1a) showed an obvious increase in dead PC3 cells from 10% (day 3) to 57% (day 5) with a combination of the precursors (iodoprazole and boronic ester) and the MR-Pd catalyst [14]. This underscored possible MR-Pd catalyzed production of the PP-121 drug and its subsequent effect of killing PC3 cells. Quantitative cell viability (Figure 1b) corroborates this observation showing a significant drop in cell viability only when the precursors are combined with the catalyst underscoring that PC3 cells are probably killed only due to in situ drug formation.Overall, the results confirm that PC3 cells death was induced only by successful in situ pro drug PP121 synthesis. The catalyst biocompatibility also means that the catalyst, on its own, cannot be the cause for PC3 cell death. The PEI in the MR-Pd catalyst structure orients the RF pre-polymer along the lines of its dendrimer structure, controlling the porosity type and extent. Porosity allows easy diffusion of the precursors, the high content of immobilized anchored Pd, evidenced in our earlier publication [13] promulgates their conversion into PP121, which then easily diffuses out mesoporous structure onto the cellular interface.In order to underscore the monolayer culture results, the mechanism of PC3 cancer cell death was studied using flow cytometry. Flow cytometry results (Figure 2a–f) showed an enhanced rate of early and late apoptosis, instead of necrosis [15].The data point density shifts from the 3rd quadrant (live cells) to the 4th quadrant (early apoptosis) followed by the 1st quadrant (late apoptosis with Annexin V and Propidium Iodide fluorescence). There is very little necrosis for both control and test groups with few data points in the 2nd quadrant. The apoptosis rate, traversing via the 4th quadrant to the 1st appears significantly more rapid with the test set (Figure 2d–f) containing both the precursors (iodoprazole and boronic ester) and MR-Pd-catalyst, compared to control (Figure 2a–c) which had Precursors without MR-Pd catalyst. This proves that PP121 anti-cancer agent was produced in situ, only when the catalyst accompanied the precursors and that it was effective in killing PC3 cancer cells.In addition, cell metabolism activity, measured by standard Presto Blue assay, also declined (Figure 2g) significantly only when the MR-Pd catalyst was introduced alongwith the precursors and not with any combination that left out either the catalyst or one of the precursors. The results, yet again, corroborated MR-Pd catalyzed cross coupling of iodopyrazole and boronic ester to PP-121 as the factor responsible for suppressing PC3 metabolic activity. The suppression of anaplastic thyroid carcinoma tumour growth by PP-121 has been reported to proceeds via mTOR-phosphatidylinositol-3-OH kinase inhibition and by binding to tyrosine kinases (VEGF receptor) [16].While the 2D model underscored the catalytic cross coupling synthesis of PP121 and its mechanism on PC3 cell death, a 3D model is a more representative evaluation for drug screening since it better simulates tissue microenvironment [17,18,19]. The use of hydrogels as a support for nano or meso structures or encapsulating material for targeted injectables is a current topin of interest, having been explored in wide ranging applications, from tissue regeneration to drug delivery [20,21]. PC3 cells were encapsulated in GeLMA—alginate hydrogel to evaluate drug screening. The two compositions used to encapsulate PC3 cells were; G5AL: GeLMA (5% w/v)-Alginate (1.5% w/v)-LAP (0.1% w/v) and G7AL: GeLMA (7% w/v)-Alginate (1.5% w/v)-LAP (0.1% w/v). The encapsulant combinations were characterized for microscopic structure, swelling and degradation (Supplementary Figure S3a–c). With increased GelMA content, porosity reduced, degradation stability increased, while the swelling ratio was comparable. Encapsulated structures were tested by cell staining, cell expansion and the Western blot tests (Supplementary Figure S3e–f). The tests underscored the cytocompatibility of the encapsulant hydrogel matrix. The screening studies also proved the necessity of the MR-Pd catalyst’s presence in producing PP121 anticancer agent, in situ, from precursors. We carried out screening studies with and without the MR-Pd catalyst. We encapsulated the PC3 cells in the G7AL composition and started the in situ drug screening 5 days after encapsulation. Additionally, we encapsulated PC3 cells in G7AL with 5 mg MR-Pd catalyst, and started the drug screening test on day 3. In both set ups, the prodrug precursors were dissolved in same medium used in the monolayer study. The catalyst was added to the well plate or injected to the encapsulated cells at day 4 and evaluation commenced on day 5.In this 3D model too, the cell death mechanism was via apoptosis rather than necrosis as evident in the flow cytometry results (Figure 3a–d).The figures underscore that the encapsulant matrix or precursors (iodopyrazole and boronic ester) without the presence of MR-Pd catalyst have little to no effect in speeding apoptosis of cancer cells (Figure 3a,b). However, when the encapsulated cells are combined with the precursors alongwith the MR-Pd catalyst, there is a rapid scale up of late apoptosis of live PC3 cells. Negligible data points in the 2nd quadrant, prove that there is no necrotic effect of either catalyst or precursors or encapsulant matrix on its own. The cytocompatibility of the encapsulant matrix and of MR-Pd, explained above, are a testament to the results. The acceleration of cells from live to late apoptosis quadrant with the precursors-catalyst combination (Figure 3c,d) underscores in situ PP-121 production pushing apoptosis. Presto blue results showed in Figure 3e show a much stronger suppression of cell expansion in the encapsulated model containing precursors-catalyst combination. In no other combinations, that left out one or more of the components (precursor or catalyst), was suppression of cellular proliferation statistically significant.The methodology employed in making of the MR-Pd catalyst used here, as per our earlier work, ensures mesoporous structure, high palladium complexing and uniform palladium distribution throughout the structure, ensuring steady conversion of precursors into PP121 [13]. We were able to encapsulate cells in a hydrogel, also containing the MR-Pd catalyst and underscore its successful application in a 3D model. Hence, a hydrogel based injectable patch can be envisaged and designed, to target tumors. The hydrogel can be loaded with the MR-Pd catalyst and precursors, which can harness the catalyst to generate steady bioavailability of the anti-cancer agent, produced under biological conditions. The transport of in situ generated PP121 agent out of the mesoporous catalyst structure in such a case could be guided by a concentration potential difference. The 3D encapsulated cellular model explored here establishes an outlook for drug screening by corroborating in situ PP-121 production under biological conditions and its efficacy in ensuring apoptosis of PC3 cells. Negligible necrosis is induced by any components. The model studied here opens up new vistas in possible on-site tumor alleviation. It can be scaled up for a practical injectable treatment modality, bypassing deleterious side effects of chemotherapy while proffering superior bioavailability, treatment efficacy at a reduced cost. In our earlier work, we also showed the application of these MR-Pd catalysts for Suzuki-Miyaura couplings in continuous microreactors, achieving conversions up to 62%. In the near future, it is possible to envisage smart applications that incorporate bio printed miniaturized, microchannel reactors with implantable pumps, made from packed beds of this MR-Pd catalysts, that can push in designated, and calculated amounts of PP121 cancer agent, produced in situ under biological conditions, bypassing cytotoxic effects on healthy neighboring tissues.