An autocatalytic multicomponent DNAzyme nanomachine for tumor-specific photothermal therapy sensitization in pancreatic cancer – Nature Communications

To build a miR-21 responsive DNAzyme system for HSP70 mRNA depletion, two common DNAzyme models (17E and 10-23) were chosen. These two DNAzymes are substrate-universal and can be tailored to recognize specific mRNAs by designing the substate arm sequence⁴². Interestingly, the 17E DNAzyme was recently found to be more active than the 10-23 DNAzyme when the same metal cofactor at the same concentration was implemented⁴³. The catalytic ring structure of the 17E DNAzyme has also been fundamentally studied (Fig. 3a, b), and the substitution of different sequence fragments in the catalytic ring can affect the catalytic activity and responsiveness of the DNAzyme to different metal ions. For instance, 3-base-pair replacements at the stem position (N³-N⁵, N⁹-N¹¹), as well as replacement of thymine (T) at the N¹² position adenine (A), can significantly enhance the Ca²⁺ affinity of the 17E DNAzyme (Fig. 3c)^32,44. Therefore, in this study, 17E DNAzyme was applied to design an efficient Ca²⁺-responsive MNAzyme dual-gene regulation machine.

a Secondary structure of the traditional 17E DNAzyme. b Variant structures of the 17E DNAzyme. N’ represents the alternative site and can be an A, T, G, or C deoxynucleotide. c Structure of the Ca²⁺-responsive 17E-based MNAzyme system. d The final MNAzyme system with high Ca²⁺ affinity for miRNA-21 triggered HSP70 mRNA cleavage. e The NUPACK web application simulated the equilibrium concentration of each binding chance. f Twenty percent polyacrylamide gel electrophoresis experiments for different formulations. g Verification of the Ca²⁺ concentration-dependent MNAzyme system. h Cancer biomarker response test. i Construction logic of the cancer biomarker-responsive MNAzyme system. The experiments in (f–h) were repeated three times independently with similar results. PTT photothermal therapy, T target strand, R right partzyme, L left partzyme, S substrate mRNA.

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We first designed the Ca²⁺-specific MNAzyme system by setting the target sequence as one complementary to miRNA-21 (abbreviated as T), while the substrate arm was engineered to match HSP70 mRNA (abbreviated as S). The MNAzyme was split from the catalytic core and divided into the right partzyme (abbreviated as R) and left partzyme (abbreviated as L) (Fig. 3d). The NUPACK web application⁴⁵ was utilized to understand the possible reaction scenarios (Fig. 3e). From the final equilibrium system of the T + R + L system (each strand concentration was set as T = 0.2 μM, R = 1 μM and L = 1 μM), T (miRNA-21) showed a strong affinity with R (right partzyme), which formed a 150 nM complex; T and L (left partzyme) showed less combination efficiency (only 3 nM). Surprisingly, T, R and L formed a 47 nM MNAzyme system (T-R-L). These simulation results unexpectedly indicated that the self-building of MNAzyme consists of two processes: T and R achieve the first conjugation, and then L combines with the T-R complex to realize the second step of MNAzyme construction. This step-by-step assembly can theoretically enhance the utilization efficiency of miRNA-21 for multiple-turnover cleavage of HSP70 mRNA in the case of low partzyme content (Supplementary Fig. 1). Similarly, the T + R + S + L reaction simulation results (L = 1 μM, R = 1 μM, T = 0.2 μM and S = 4 μM) showed that the T-R-L system bound to the substrate S sequence and formed a 44 nM (T-R-S-L) complex. Of note, the T-R-S-L complex exhibited a lower free energy of secondary structure (−38.96 kcal/mol) compared with that of the T-R-L system (−27.84 kcal/mol), which revealed that the T-R-S-L structure was more stable (Supplementary Fig. 1).

Then, the feasibility of MNAzyme-based dual-gene modulation was evaluated in reality by 20% polyacrylamide gel electrophoresis (PAGE) tests (Fig. 3f). The individual L, R, S and T strands all showed clear bands (Lanes 1, 2, 3 and 4), while an obvious new band appeared upon incubation of L, R and T for 30 min at 37°C (L = 1 μM; R = 1 μM; T = 0.2 μM, Lane 5). These results indicated that partzymes possessed strong binding affinity with miRNA-21, which facilitated efficient MNAzyme construction. For a deeper understanding of the multiple turnover catalytic cleavage effect of the MNAzyme system on the HSP70 substrate strand (S), we incubated excess S chain (4 μM) with the abovementioned system, as revealed in Lane 6. A large amount of unreacted S strand appeared at the top of the band (marked with a red asterisk in Fig. 3f, Lane 6). Importantly, the S strand was not cleaved due to the lack of catalysis by the metal ions in Lane 6. Subsequently, when 10 mM Ca²⁺ was added to the system in Lane 7, the substrate chain S was cleaved and disappeared, and a new band was formed from the PAGE test result, as shown in Fig. 3f, Lane 7. These results demonstrated that our designed partzymes could utilize 0.2 μM miRNA to achieve Ca²⁺-mediated 4 μM HSP70 multiple-turnover cleavage and consume all miRNA-21 to achieve dual gene regulation.

To further verify the metal ion dependence of the partzyme system, a substrate catalytic hydrolysis test was performed under different Ca²⁺ concentrations for 4 h (Fig. 3g). L (1 μM) and R (1 μM) strands together with 0.2 μM T strand were applied. We found that as the Ca²⁺ concentration increased from 0 to 50 mM, the content of substrate S (4 μM) gradually decreased, and the product amount consistently increased. Furthermore, the hydrolysis rate of substrate strand HSP70 mRNA reached saturation at ~20 mM Ca²⁺. These results reflected that a high catalyst rate of the MNAzyme system could be guaranteed when sufficient calcium ions were present in the mRNA regulation system.

Ultimately, to demonstrate the cancer responsiveness of the designed partzymes, i.e., their safety in healthy tissues, we carried out another PAGE assessment to investigate the hydrolysis performance of the MNAzyme system for HSP70 mRNA in the absence of miRNA-21 (with 50 mM Ca²⁺) (Fig. 3h). We observed that although in the presence of sufficient Ca²⁺, partzymes could not self-assemble into MNAzymes to mediate the catalytic hydrolysis of HSP70 mRNA. Therefore, hypothetically, when partzymes, Ca²⁺ and a photothermal therapeutic agent (IR780) are simultaneously delivered to the tumor microenvironment (TME), the partzymes can distinctively self-assemble into an MNAzyme-driven HSP70 mRNA-regulating machine in specific miRNA-21-overexpressing pancreatic cancer cells. These processes avoid hysteresis effects by preventing the generation of heat shock proteins at the mRNA level before photothermal treatment. Moreover, during the assembly process of MNAzymes, most of the miRNA-21 within the TME will be consumed, thus realizing dual-gene regulation for both the pathogenic gene miRNA-21 and the therapeutic stimulated gene HSP70 (Fig. 3i).

The miRNA-21-dependent self-construction process of MNAzyme and the specific gene cleaving function under Ca²⁺ triggering have been verified, yet the intracellular activation of the MNAzyme system suffers from poor cellular internalization and low cytoplasmic Ca²⁺ content (~0.1 μM)⁴⁶. Therefore, a smart and robust partzyme delivery strategy using Ca²⁺ as the multicargo carrier component to achieve target site Ca²⁺ enhancement has been explored. Nanostructured amorphous calcium phosphate (CaP) has been broadly investigated as an excellent nuclear acid delivery system owing to its high affinity for the gene phosphate backbone (Fig. 4a)⁴⁷, pH-responsive degradability and sustained release performance⁴⁸. However, amorphous calcium phosphate (ACP) composed of Posner’s clusters (Ca₉(PO₄)₆) is an unstable precursor that can dissolve and renucleate as hydroxyapatite (HA) within a few minutes in aqueous solution^49,50, causing inefficient cargo delivery. To verify this phenomenon, transmission electron microscopy (TEM) was utilized to systematically observe the crystal transformation of ACP, as shown in Fig. 4b. We found that the ACP was a complete and smooth sphere with irregular channels on its surface within 10 min of preparation. However, the storage of APC in Milli-Q water resulted in the dissolution of ACP (>1 h), nucleation (>3 h) and full crystallization to HA after 48 h. Moreover, we also found that this crystal transformation process could take place in the cell culture medium after 24 h of incubation and that the large size and irregular needle-like shape structure made it difficult to deliver nucleotide drugs and even calcium ions into cells (Fig. 4c).

a Schematic illustration of the DNA and CaP biomineralization process. b Degradation process of CaP in Milli-Q aqueous solution. c Panc-1 cells were incubated with APC nanoparticles for 24 h. d Stability for Zn-substituted CaP in Milli-Q aqueous solution. e Interaction between PEG and Ca²⁺ and Zn²⁺. Carbon, oxygen, and hydrogen atoms are labeled with black, red, and white, respectively. f TEM images of ^RM_(I+C) and ^RM_(I+C)@CaP_(p). g Size distribution of ^RM_(I+C)@CaP_(p) NPs within 2 weeks and zeta potential results for ^RM_(I+C), Zn-doped CaP and ^RM_(I+C)@CaP_(p) (n = 3 independent experiments, and the data are presented as the mean values ± SDs). h FTIR results for Zn-substituted CaP and ^RM@CaP. i Photothermal heating curves of ^RM_(I+C)@CaP_(p) NPs with different concentrations of IR780. j The infrared thermal images correspond with photothermal heating curves. k Temperature elevation of ^RM_(I+C)@CaP_(p) NPs over four rounds of NIR on–off irradiation cycles. l Photobleaching effects of pure drug and nanoparticles after three rounds of laser irradiation (n = 3 independent experiments, and the data are presented as the mean values ± SDs). m Drug-releasing capacity of ^RM_(I+C)@CaP_(p) under different situations (n = 3 independent experiments and the data are presented as the mean values ± SDs). n Cleavage capacity of HSP70 mRNA after partzyme release from different environments. All statistics were calculated using two-tailed paired t tests, and the experiments in (b–d, f, n) were repeated three times independently with similar results. The source data from (g, h, I, k, i, m) are provided as a Source Data file.

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The zinc additive was found to stabilize the ACP phase for more than 20 days when the Zn/Ca ratio was 10%⁵¹. In the reaction system in which Zn²⁺ and Ca²⁺ existed simultaneously, the chemical reactions followed the equations below⁵²:

In this way, highly stable ACP NPs were fabricated, as depicted in Fig. 4d, which contributed to the mechanism by which smaller zinc ions can easily replace Ca²⁺ in ACP and inhibit crystallization by distorting atomic order and reducing ACP solubility⁵¹. To assess the content of Ca²⁺ and DNAzymes, we used two different assay kits to measure the concentrations of Ca²⁺ and Zn²⁺ ions within the nanosystem, and we found the concentration of zinc ions to be 196 μg and the concentration of calcium ions to be 1280 μg within 3.5 mg Zn-doped CaP NPs.

Nevertheless, Zn²⁺-substituted APC NPs did not possess cancer cell-enhanced endocytosis, and surface pores were reduced by the tight cooperation between zinc and phosphate ions, resulting in the inability to load multiple therapeutics. To resolve the abovementioned difficulties, RGD-modified DSPE-PEG amphiphilic micelles (^RM) were used as the synthesis template of Zn²⁺-doped CaP since the negatively charged oxygen atom of the CH₂-O-CH₂ group in PEG can attract Ca²⁺ or Zn²⁺ through electrostatic attraction (Fig. 4e)⁵³. Meanwhile, the micelles can be loaded with multiple hydrophobic drugs, and RGD can realize cancer cell targeting⁵⁴.

We first synthesized IR780/Cur-loaded micelles, and a spherical structure of ~50 nm could be seen under TEM (Fig. 4f, ^RM_(I+C)). During preparation, we found that 50 mg of DSPE-PEG-RGD could carry up to 7.98 mg of IR780 and 2.06 mg of Cur. (Tables S1 and S2). To achieve the optimal synergistic treatment ratio of both drugs after being loaded by the nanosystem, the loading weight ratio of IR780:Cur (mg) was adjusted to 0:2, 0.5:2, 1:2, 2:2, 4:2, and 8:2 to calculate the coefficient of drug interaction (CDI)^55,56. The calculation was based on the WST-1 absorbance results, which compared the viability of cells after treatment with different drug ratios of nanoparticles. The equation is as follows:

where AB is the absorbance of the combination groups to the control group, while A or B is the absorbance of the single-agent group to the control group (CDI > 1, =1, <1 indicates antagonism, additivity and synergism). In accordance with the WST-1 results, a 4:2 ratio (4 mg IR780 and 2 mg Cur within 50 mg DSPE-PEG-RGD) exhibited the best CDI = 0.52 (Tables S3 and S4).

After attaining the proper drug loading, we further determined the optimum dose of partzyme, and PO₄²⁻, Zn²⁺ and Ca²⁺ were utilized to mineralize partzyme (P), as shown in Fig. 4f, ^RM_(I+C)@CaP_(p). For silencing miRNA-21 in pancreatic cancer cell lines, it has been reported that the concentration of miRNA-21 antisense oligonucleotides needs to reach 100 nM to achieve a significant gene-silencing effect^57,58. Hence, the therapeutic concentration of partzyme, which also serves as a miRNA silencing agent, should also reach 100 nM. To confirm the efficient partzyme loading content of our designed nanosystem, we carried out a gradient partzyme loading test (Supplementary Fig. 2). Left and right partzymes were labeled with Cy3 and mixed with 1 ml ^RM_(I+C) solution (one/tenth upper-mentioned micelle volume, which contained 0.4 mg IR780 and 0.2 mg Cur) to yield different concentrations of each partzyme (1, 10, 50, 100, and 200 μM). After calcium phosphate mineralization, we found that all partzymes were centrifuged to the bottom of the tube (even when the content was as high as 200 μM), and there was no leakage of partzyme after several washes with Milli-Q water. Therefore, the system had excellent partzyme encapsulation efficiency and eliminated the burst release phenomenon of oligonucleotides through a rational biomineralization process to easily achieve a 100 nM therapeutic concentration.

To further demonstrate the successful synthesis of ^RM_(I+C), CaP_(p) and ^RM_(I+C)@CaP_(p), dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR) were used to characterize the particle size, zeta potential and characteristic chemical bond of the nanocarrier. The particle size analysis revealed that ^RM_(I+C) possessed a similar average diameter as ^RM_(I+C)@CaP_(p), at ~100 ± 15 nm (Supplementary Fig. 3), which was larger than the TEM results (~50 nm) due to the aqueous layer out of the DLS specimen⁵⁹. Notably, ^RM_(I+C)@CaP_(p) was found to be very stable (with the same size) in aqueous solution for more than 2 weeks (Fig. 4g), suggesting that this nanosystem possessed excellent stability, which may have occurred through a combination of zinc-additive and steric repulsion forces between the PEG chains. Additionally, in PBS buffer at pH = 7.4 (Fig. 4g), ^RM_(I+C) was observed to have a zeta potential of −10.21 mV, which may have been attributable to the carboxyl groups on RGD. Furthermore, the Zn²⁺-substituted CaP_(p) showed a strong positive charge (10.01 mV). Through the elemental mapping experiment, we found that the Zn ions and calcium ions were distributed throughout the NPs; hence, the positive charge may have been due to the uncoordinated calcium and zinc ions on its surface (Supplementary Fig. 4). Finally, we found that the final formulation ^RM_(I+C)@CaP_(p) exhibited −2.53 mV, which might have been caused by the outermost exposed RGD that renders the formulation slightly electronegative. Based on the aforementioned results, we conducted stability assessments on the final formulation of our nanomaterial in accordance with the guidelines provided by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Supplementary Table S5. We investigated the stability of the formulation at refrigerator temperature (4–8 °C) and under accelerated conditions at 40 °C, by following with the ICH guidelines, specifically ICH Q1A(R2) for long-term stability testing. The results of these stability studies indicated that there was no significant increase (p > 0.05) in particle size or polydispersity index (PDI) throughout the 3-month experimental period.

The FT-IR spectrum of DSPE-PEG-COOH reflected an absorbance band at 1738 cm⁻¹, corresponding to the –COOH groups⁶⁰. After RGD modification, there was a newly formed band at 1657 cm⁻¹ belonging to the O=C-N-H group between PEG and RGD connections within micelles⁶⁰. Subsequently, the micelles were coated by CaP, and the band at 1657 cm⁻¹ was also found in the ^RM_(I+C)@CaP group compared with the pure CaP group. These results together demonstrated the successful incorporation of CaP nanoparticles with the DSPE-PEG-RGD micelles (Fig. 4h).

IR780, as a PTT and PDT agent, suffers from poor aqueous solubility and high photobleaching properties and was protected inside micelles in this study for higher drug loading content and enhanced therapeutic effects. To examine the photothermal conversion properties of the final formulation, ^RM_(I+C)@CaP_(p), which contains different concentrations of IR780 (0, 2, 5, 50, 100 μg/ml), was irradiated with an 808 nm laser (1 W cm⁻²) for 300 s (Fig. 4i). The corresponding infrared photos are shown in Fig. 4j. The results showed that the photothermal conversion properties of ^RM_(I+C)@CaP_(p) displayed a positive correlation with the NP concentration. The solution temperature increased by up to 20 °C when only 5 μg/ml IR780 NPs were irradiated for 5 min (Fig. 4i, j), while pure water did not increase the temperature.

Furthermore, the photothermal conversion efficiency (η) of pure IR780, ^RM_(I+C) and ^RM_(I+C)@CaP_(p) NPs was also studied through NIR heating-cooling cycles (1.0 W cm⁻², 808 nm) with 50 μg/ml IR780 (Fig. 4k). (η) was calculated by following Eqs. (5) and (6)⁵⁹:

Here, ΔT_max is the maximum temperature change value, while Q is the solvent increased heat, I is the power of the laser, A⁸⁰⁸ is the absorbance of the NPs at a wavelength of 808 nm, and hs is defined by another formula:

in which m and c are the specific heat capacity and mass of the solution, respectively, while τ is the slope of the fitted line of time to (-{{{{mathrm{ln}}}}}frac{Delta T}{{Delta T}_{max }}).

The photothermal conversion efficiency of the pure drug was only 23.70% (Supplementary Fig. 5) due to the poor stability of IR780 in aqueous solution. In contrast, the η of ^RM_(I+C) was converted up to 29.25% (Supplementary Fig. 6), which demonstrated that the micelle could prevent IR780 degradation. Surprisingly, the Zn²⁺-containing dense CaP mineralization layer further improved the η of IR780 (33.70%, Supplementary Fig. 7), indicating that the final formulation significantly improved the photothermal stability of IR780. Furthermore, the photobleaching efficiency was additionally evaluated for the abovementioned IR780, ^RM_(I+C) and ^RM_(I+C)@CaP_(p) groups (Fig. 4l). After three cycles of irradiation (1 W cm⁻², 5 min, 808 nm), free IR780 showed an ~80% decrease in absorbance at 780 nm, illustrating that free IR780 was rapidly degraded. Conversely, ^RM_(I+C) reflected a 41% decrease in absorbance, and ^RM_(I+C)@CaP_(p) showed only a 23% decrease after 3 cycles of irradiation. Hence, compared with ^RM_(I+C), ^RM_(I+C)@CaP_(p) realized an effective photothermal conversion ability of IR780.

To further estimate the pH-responsive drug release capacity, ^RM_(I+C)@CaP_(p) NPs (containing 0.4 mg of IR780, 0.2 mg of Cur and 200 μM partzyme) were subjected to release tests by dialysis membranes. Since IR780 generates heat under laser irradiation, it will promote the diffusion of drug molecules from the material, so different drug release environments with pH and temperature were designed (pH = 7, 37 °C; pH = 7, 45 °C; pH = 6, 37 °C; pH = 6, 45 °C). Therapeutic agents were observed through UV–visible spectroscopy at 780, 425 and 560 nm (Supplementary Fig. 8). We observed that at 45 °C, there was an increased release of the drug. Approximately 35% IR780 and 43% Cur were released at pH = 6 and 45 °C, whereas only 9% IR780 and 14% Cur were released at pH = 6 and 37 °C. A similar phenomenon was also found in the pH = 7 group. More than 20% IR780 and 30% curcumin were released at pH = 7 and 45 °C. However, less than 5% of IR780 and 10% of curcumin were released at pH = 7 at 37 °C.

Furthermore, we observed a pronounced pH-responsive release profile for partzymes since CaP possessed excellent pH-sensitive degradation performance, and more than 90% of the partzyme was released from the system after 4 h incubation at pH = 6 (Fig. 4m, partzyme). However, the degradation of CaP did not mediate significant release of Cur and IR780 at pH 6 compared to partzymes loaded in the CaP shell, reflecting that IR780 and Cur were loaded mainly inside the micelles. Subsequently, 1 μl of released medium from each group was further incubated with 1 μl of miRNA-21 (10 μM) and 2 μl of HSP70 mRNA (10 μM). We found that the released media in the pH = 6 groups efficiently cleaved HSP70 (Fig. 4n). Rapid release of partzymes ensured silencing of HSP70 before PTT treatment and prevented the previously mentioned hysteretic effect.

Given the successful design and construction of the dual-gene regulation MNAzyme system and the productive fabrication of the ^RM_(I+C)@CaP_(p) nanovector with high partzyme loading and excellent aqueous stability, we next closely investigated the biocompatibility of the carrier system by testing the toxicity of the pure nanocarrier and laser to the normal human dermal fibroblast NFDH cell line (Supplementary Fig. 9). We found that pure nanovehicle ^RM@CaP at a maximum concentration of 40.5 μg/ml (which could carry 6 μg/ml IR780 and 3 μg/ml curcumin) was not toxic to healthy cells. Hence, based on the results in Supplementary Table S3, at effective therapeutic concentrations for PANC-1 cells (IR780: 4 μg/ml, Cur: 2 μg/ml), pure carriers should have negligible side effects. In addition, NFDH cells were irradiated with an 808 nm laser at different laser intensities (0, 0.5, 1, 1.5 and 2 W cm⁻²), and it was found that a pure laser did not inhibit the growth of healthy cells (Supplementary Fig. 9). These results suggest that the ^RM@CaP system holds good biocompatibility and that the laser power we selected is safe for healthy cells.

Next, the cellular uptake of the pure drug, M_(I+C)@CaP_(p) and ^RM_(I+C)@CaP_(p) groups by PANC-1 cells and the healthy control cell line NHDF (after 1 h and 8 h incubation) was observed by tracking the fluorescence of IR780 (red), Cy3-modified partzymes (green) and Cur (light blue) via confocal laser scanning microscopy (CLSM) and flow cytometry (Fig. 5a and Supplementary Figs. 10–12). The therapeutic concentrations were as follows: IR780: 4 μg/ml, Cur: 2 μg/ml and partzyme: 2 μM. In the case of PANC-1 cells, the results revealed that the uptake content of each therapeutic agent gradually increased with the extension of the incubation time (Supplementary Figs. 10 and 5a). After 8 h of incubation with the pure drug group, IR780 and Cur were pronounced diffused into PANC-1 cells, while Cy3-labeled partzyme exhibited no internalization. However, for the M_(I+C)@CaP_(p) group, IR780, Cur and Cy3-labeled partzyme were all endocytosed, which proved that the nanocarrier could ensure the internalization of partzyme. Meanwhile, after the decoration of RGD on top of the nanosystem, the ^RM_(I+C)@CaP_(p) group exhibited 2.2 times higher uptake efficiency than the M_(I+C)@CaP_(p) group, which reflected that RGD, as a tumor-targeting tripeptide, could promote the endocytosis of NPs by pancreatic cancer PANC-1 cells. Inductively coupled plasma‒optical emission spectrometry (ICP‒OES) results indicated that the calcium ion concentration in the cancer cells was ~240 parts per million (ppm), which was sufficient to activate MNAzymes (Supplementary Fig. 10).

a Uptake of pure drug, M_(I+C)@CaP_(p) and ^RM_(I+C)@CaP_(p) groups by PANC-1 cells (n = 3 independent experiments, and the data are presented as the mean values ± SDs). b Lysosomal escape profiles of ^RM_(I+C)@CaP_(p) groups (±Laser) by PANC-1 cells at 2 h and 24 h. The Pearson value Rcoloc was calculated by using ImageJ with the Coloc 2 plugin. c The schematic diagram depicts the intracellular self-assembly of the MNAzyme system for regulating the protein expression of PTEN and HSP70. d Intracellular miRNA-21 content was determined by the fluorescence in situ hybridization (FISH) method (n = 3 independent experiments and the data are presented as the mean values ± SDs). e The expression of HSP70 and PTEN protein was evaluated by western blotting. The final concentrations of the components in the different groups were as follows: IR780: 4 μg/ml, Cur: 2 μg/ml, each partzyme: 2 μM) (n = 3 independent experiments, and the data are presented as the mean values ± SDs). Groups 1–8 represent the following: 1: PBS, 2: PBS +Laser, 3: I + C, 4: I + C +Laser, 5: ^RM_(I+C)@CaP, 6: ^RM_(I+C)@CaP +Laser, 7: ^RM_(I+C)@CaP_(p), and 8: ^RM_(I+C)@CaP_(p) +Laser. All statistics were calculated using two-tailed paired t tests, and the experiments in (a, b, d, e) were repeated three times independently with similar results. The source data from (a, d, e) are provided as a Source Data file.

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In contrast, for NHDF cells, the pure drug, M_(I+C)@CaP_(p) and ^RM_(I+C)@CaP_(p) groups all showed negligible uptake after 1 h of treatment (Supplementary Fig. 11). Meanwhile, only sporadic drug fluorescence signals were exhibited for the M_(I+C)@CaP_(p) and ^RM_(I+C)@CaP_(p) groups after 8 h of incubation (Supplementary Fig. 12). These phenomena may be attributable to the weaker uptake capacities of normal cells⁴¹. The surface modification of RGD did not promote the ingestion of NPs by healthy NHDFs.

Having determined that the ^RM_(I+C)@CaP_(p) group could effectively deliver multiple cargoes into PANC-1 cells, we explored the intracellular endosomal escape. For both groups, after NPs and cells were cultured together for 2 h and 24 h, the cell media were replaced, and a 1 W laser was applied for 5 min. Here, IR780 is presented in red, and the lysosome is labeled in green. Pearson correlation coefficients (PCCs) were utilized to determine whether the two colors colocalized (>0.5 indicates moderate correlation; <0.5 indicates low correlation)^61,62. After 2 h of treatment with ^RM_(I+C)@CaP_(p) NPs without laser irradiation, we found that the red fluorescence (IR780) overlapped well with the green fluorescence of lysosomes (Fig. 5b). In addition, the PCC was calculated as 0.673 (higher than 0.5), demonstrating the colocalization of IR780 and lysosomes. After 1 W cm⁻² 808 nm laser irradiation for 5 min, the PCC number dropped to 0.582 but still showed a colocalized effect. For the ^RM_(I+C)@CaP_(p) NPs after 24 h of treatment, lower overlap between IR780 and lysosomes was observed, and the PCC value was 0.454. Notably, there was almost no overlap between red and green fluorescence after laser irradiation (PCC = 0.413), confirming that the therapeutic agents could efficiently escape from the lysosome.

After validating that the therapeutics could be efficiently delivered to the cytoplasm, we further studied the functional performance of each nanosystem component (MNAzyme, IR780, Cur and Ca²⁺) in PANC-1 cells. For the MNAzyme system, self-assembly into MNAzyme should be achieved by the released partzymes and endogenously overexpressed miRNA-21, which then autocatalytically trigger its substrate (HSP70 mRNA) cleavage function with the assistance of Ca²⁺ (Fig. 5c). Subsequently, the silencing of miRNA-21 can mediate the increased expression of the tumor suppressor protein PTEN, while the HSP70 protein should be downregulated after HSP70 mRNA cleavage.

Based on the above assumptions, to verify that the partzymes could bind to intracellular miRNA-21, the fluorescence in situ hybridization (FISH) method was used to observe intracellular free miRNA-21 (Fig. 5d). Four groups, including the PBS, IR780+Cur, ^RM_(I+C)@CaP (without partzymes) and ^RM_(I+C)@CaP_(p) (with partzymes) groups, were used, and endogenous miRNA-21 was labeled red. From the results, we observed that the PBS groups exhibited tremendous Cy5.5 fluorescence (miRNA), while when IR780 and curcumin were added, the fluorescence intensity of miRNA decreased by nearly 32%. It has been reported that curcumin can regulate a variety of miRNAs (including miRNA-21) in other cancer cell lines⁶³. Hence, curcumin could also inhibit endogenous miRNA-21 in the PANC-1 cell line. Of note, during the experiments, all the therapeutics containing cell culture medium were replaced at the 6 h timepoint, which resulted in a shorter intracellular retention time for the pure drug, while the nanosystem promoted the retention time and therapeutic efficiency of Cur. As a result, the red fluorescence intensity in the ^RM_(I+C)@CaP group was decreased by ~76%. More interestingly, from the results of the ^RM_(I+C)@CaP_(p) group, the red fluorescence intensity was reduced almost to zero, indicating that the partzyme system could efficiently utilize miRNA-21 and ensure MNAzyme formation.

Subsequently, to explore whether MNAzyme can effectively downregulate the expression of HSP70 protein and mediate the increase in the expression of PTEN protein in PANC-1 cells, western blot experiments were performed, and the relative HSP70/GAPDH and PTEN/GAPDH ratios were calculated with ImageJ, as shown in Fig. 5e. For the HSP70 protein, compared with | PBS (1 and 2), we found that IR780+Cur with laser treatment (4) significantly upregulated HSP70 expression (1.8 times), which indicates that PTT can activate HSP70 to protect cells from thermal stress⁶⁴. Surprisingly, for the ^RM_(I+C)@CaP groups (5 and 6), the expression of HSP70 was downregulated by nearly 40% (-laser) and 20% (+laser). It has been reported that the generation of HSPs highly depends on ATP production^8,9. Since our nanosystem will trigger multichannel Ca²⁺ ions inside cells to cause mitochondrial calcium overload (Figs. 6 and 7), ATP production by damaged mitochondria should decline and lead to a decrease in HSP70 content. Ultimately, from the results of the ^RM_(I+C)@CaP_(p) groups with partzymes 7 and 8, the HSP70 gene was fully eliminated for both the ±laser groups, suggesting the effective HSP70 mRNA cleavage function of the MNAzyme system.

a Colocalization between IR780 and mitochondria. b ¹O₂ generation capability of each treatment group (n = 3 independent experiments, and the data are presented as the mean values ± SDs). c Ca²⁺ concentration level visualization by using Fluo-3 AM as a labeling dye (n = 3 independent experiments and the data are presented as the mean values ± SDs). d ΔΨm detection by tracking of the JC-1 monomer and aggregates through CLSM. e Relative fluorescence intensity of JC-1 signals for each group as calculated by ImageJ. The eight groups are labeled from 1 to 8, including PBS (1), PBS +Laser (2), IR780+Cur (3), IR780+Cur +Laser (4), M_(I+C)@CaP_(p) (5), M_(I+C)@CaP_(p) +Laser (6), ^RM_(I+C)@CaP_(p) (7) and ^RM_(I+C)@CaP_(p) +Laser (8). The final concentrations of the components in the different groups were as follows: IR780: 4 μg/ml, Cur: 2 μg/ml, and each partzyme: 2 μM (n = 3 independent experiments, and the data are presented as the mean values ± SDs). All statistics were calculated using two-tailed paired t tests, and the experiments in (a–d) were repeated three times independently with similar results. The source data from (b, c, e) are provided as a Source Data file.

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a Bio-TEM image for mitochondrial morphology observation. b Western blot experiments for apoptosis-related protein detection. Eight groups (1–8) were tested, including the PBS, PBS +Laser, I + C, I + C +Laser, ^RM_(I+C)@CaP, ^RM_(I+C)@CaP +Laser, ^RM_(I+C)@CaP_(p) and ^RM_(I+C)@CaP_(p) +Laser groups. (n = 3 independent experiments). c The relative ratio of each protein was calculated with ImageJ (n = 3 independent experiments, and the data are presented as the mean values ± SDs). d Apoptosis analysis of PANC-1 cells under Annexin V-APC/PI staining_. (mP means mutated partzyme, which can only target miRNA but cannot achieve HSP70 silence; all groups were under laser irradiation). All statistics were calculated using two-tailed paired t tests, and all experiments (a, b, d) were repeated three times independently with similar results. The source data from (b, c) are provided as a Source Data file.

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PTEN protein expression should be upregulated owing to the silencing of miRNA-21. From the results, compared with the PBS groups (1 and 2), the pure drug groups (3 and 4) showed slight upregulation of PTEN expression due to the inhibitory effect of curcumin. Meanwhile, the relative ratio of PTEN/GAPDH for the ^RM_(I+C)@CaP groups (5 and 6) was ~2 times higher than that of the PBS groups, which might have been caused by the long-term Cur-mediated decrease in miRNA-21. Furthermore, for the ^RM_(I+C)@CaP_(p) groups (7 and 8), a significant increase in the PTEN/GAPDH relative ratio (3 times higher than that of the PBS groups) was observed, demonstrating the miRNA-21 silencing effect of the MNAzyme system.

Considering that the MNAzyme system has a targeted therapeutic effect on cancer cells, we further investigated MNAzyme function in healthy NFDH cells in eight groups. The FISH method was first utilized to compare the miRNA-21 content between NFDH cells and PANC-1 cells (Supplementary Fig. 13). The green fluorescence in NFDH cells was clearly ~7 times weaker than that in PANC-1 cells, indicating that partzymes might not effectively use miRNA-21 to construct the MNAzyme system. Moreover, due to the absence of RGD receptors on the surfaces of healthy cells, the construction of the MNAzyme system was hampered by both poor partzyme delivery efficiency and the absence of miRNA-21, which could not further regulate the expression of HSP70 proteins. (Supplementary Fig. 14). Western blotting was then applied to test the generation of HSP70 in NFDH cells. The results showed that IR780 + Cur + laser (4) increased HSP70 expression by 2 times compared with that in the PBS group, while ^RM_(I+C)@CaP + laser (6) and ^RM_(I+C)@CaP_(p) + laser (8) increased the HSP70/GAPDH relative ratio by 3.4 times (Supplementary Fig. 14), demonstrating that the nanosystem did not affect the thermal protection mechanism of healthy cells.

After confirming the cancer-specific dual-gene regulatory capability of the MNAzyme system, we further studied subcellular-based ion therapy with our nanoplatform in PANC-1 cells. Multidimensional mitochondrial therapeutics was realized through the following synergistic mechanisms: (1) IR780 targeted mitochondria to generate PTT and PDT therapy; (2) CaP-composed nanocarriers released tremendous amounts of Ca²⁺, mediating intramitochondrial Ca²⁺ overload; and (3) Cur further promoted the release of Ca²⁺ from the ER and inhibited Ca²⁺ efflux pumps in cancer cells.

We first verified the mitochondria-targeted therapy with IR780 through CLSM, as shown in Fig. 6a. Eight groups, including PBS ±Laser, I + C ±Laser, M_(I+C)@CaP_(p) (without RGD) ±Laser and ^RM_(I+C)@CaP_(p) (with RGD) ±Laser, were established and marked as 1 to 8. The laser groups were under 1 W cm⁻² 808 nm irradiation for 5 min. IR780 exhibited red fluorescence, and mitochondria were marked in green. As expected, IR780 colocalized with mitochondria in each treatment group. Compared with those of the PBS and pure drug groups (groups 1–4), the green fluorescence signals of the M_(I+C)@CaP_(p) group without laser (5) were decreased, which might have been caused by Ca²⁺-mediated mitochondria disruption. M_(I+C)@CaP_(p) +Laser (6) showed less green signal than (5) due to the synergistic effect of IR780 and calcium overload. In contrast, after modification with RGD, the ^RM_(I+C)@CaP_(p) ±Laser group (7 and 8) exhibited the least green fluorescence, indicating the most severe mitochondrial damage caused by RGD-mediated enhanced endocytosis of the nanosystem by cancer cells.

In addition, the intracellular ¹O₂ generation capability was also monitored to investigate the PDT effect of IR780. The same groups mentioned above were implemented, and the ROS were tracked with 2′,7′-dichlorofluorescein diacetate (which emits a green signal after oxidation by ¹O₂), as depicted in Fig. 6b. All +Laser treatment groups produced observable ¹O₂ upon laser irradiation for 5 min (808 nm 1.0 W cm⁻²)_. We found that the pure drug I + C +Laser group (4) showed less relative fluorescence intensity, which was due to poor drug stabilization and solubility in the cell culture medium. The ^RM_(I+C)@CaP_(p) +Laser group (8) produced the strongest green signal; comparatively, only 75% of the relative fluorescence intensity in the M_(I+C)@CaP_(p) +Laser group (6) was found, which was calculated by ImageJ, reflecting the enhanced therapeutic effect mediated by RGD decoration.

Later, Ca²⁺-based ion therapy was further studied by using Fluo-3 AM (green fluorescence) to visualize intracellular Ca²⁺ via CLSM, and the relative intensity of Ca²⁺ was calculated with ImageJ (Fig. 6c). The same groups were selected, and an 808 nm 1 W cm⁻² laser was chosen for 5 min. As expected, a noticeable increase in Ca²⁺ concentration was found in the ^RM_(I+C)@CaP_(p) ±Laser groups (7 and 8), indicating the high efficiency of our designed nanoplatform for inducing mitochondrial Ca²⁺ overload. Meanwhile, compared with M_(I+C)@CaP_(p) ±Laser (5 and 6), Groups 7 and 8 showed 2 times higher Ca²⁺ relative intensity levels, and the increases were attributable to the RGD-mediated enhancement of the endocytosis effect. In addition, with the assistance of curcumin, the ^RM_(I+C)@CaP_(p) group showed an excellent plasma membrane calcium pump (PMCA ATPase) silencing effect and achieved higher Ca²⁺ levels than the group without Cur (Supplementary Fig. 15), indicating the synergistic function of curcumin and Ca²⁺-containing nanocarriers.

Mitochondrial transmembrane potential (ΔΨm) represents the hyperpolarization/depolarization of mitochondria. To ascertain that the mitochondria experienced a disruption effect from our designed nanosystem, JC-1 dye was further employed as a sensor (JC-1 aggregates exhibit a red signal for healthy cells with high ΔΨm, and the JC-1 monomer exhibits a green signal for damaged cells with low ΔΨm). The same groups were used as elucidated in Fig. 6d, and the relative signal intensities of aggregates and monomers were calculated by ImageJ (Fig. 6e). After laser irradiation for 5 min (808 nm 1.0 W cm⁻²), compared with the PBS group (1 and 2), the I + C +Laser group (4) showed 1.5 times higher green fluorescence, suggesting a decrease in ΔΨm. In contrast, the 2-fold higher monomer content in the M_(I+C)@CaP_(p) +Laser group (6) suggested synergistic mitochondrial damage by IR780 and Ca²⁺ overload. Surprisingly, with RGD decoration, the ^RM_(I+C)@CaP_(p) ±Laser group (7 and 8) showed 3.16 and 3.6 times higher monomer signals, respectively, indicating that RGD modification significantly enhanced the therapeutic efficiency of the pure drug and efficiently mitigated mitochondrial dysfunction by inducing the synergistic effect of PDT/PTT and Ca²⁺ overload.

Later, to directly observe the mitochondrial morphology changes, bio-TEM was used to analyze each aforesaid group, and the inclusions inside mitochondria were marked with a red star (Fig. 7a). In general, a higher energy requirement for cancer cells results in more mitochondria with abundant cristae. As expected, we found tremendous amounts of mitochondria with dense cristae in PANC-1 cells in the PBS ± Laser groups. In addition, mild mitochondrial destruction (slight swelling) was observed in the I + C +Laser groups, while the therapeutic effect was not prominent. Of note, noticeable mitochondrial swelling and cavitation effects were detected in the M_(I+C)@CaP_(p) +Laser groups, which proved that the nanosystem could significantly enhance mitochondrial dysfunction through IR780 and superabundant calcium. It is worth mentioning that with RGD modification, negligible cristae were found in the ^RM_(I+C)@CaP_(p) group, suggesting that RGD mediated enhanced curative efficiency.

MNAzyme-mediated enhanced photothermal therapy and Ca²⁺ overload treatment increased mitochondrial permeability, resulting in the release of Cyto-C and finally activating Caspase-3 to mediate apoptosis. The apoptosis mechanism was investigated through western blot experiments (Fig. 7b), and the relative ratio of each protein was calculated by ImageJ (Fig. 7c). From the results, compared with the PBS group (1 and 2), the I + C +Laser group (4) showed a slight increase in Cyto-C, which reflected inefficient damage to mitochondria. In contrast, the ^RM_(I+C)@CaP +Laser group (6) showed ~1.5 times higher Cyto-C release than the I + C +Laser group (4), which was related to enhanced mitochondrial calcium overload. Importantly, due to the MNAzyme-mediated PTT-enhancing effect, the ^RM_(I+C)@CaP_(p) +Laser treatment (8) greatly led to the release of Cyto-C from mitochondria into the cytoplasm (2.5 times higher than that in the PBS group), which mediated pro-caspase cleavage, and cleaved caspase-3 was found at up to 3 times higher levels than that in the PBS group, as shown in Fig. 7c. Therefore, the ^RM_(I+C)@CaP_(p) nanosystem efficiently upregulated apoptosis-related proteins, resulting in significant apoptosis.

Based on the aforementioned experiments, we successfully validated the upregulation effect of the nanosystem on PTEN, the silencing effect on HSP70, and the calcification effect on mitochondria. Given ample evidence that these rationally designed nanoformulations can achieve excellent cancer therapy, we further investigated their multiple synergistic effects through WST-1 and flow cytometry for both the NHDF and PANC-1 cell lines (Supplementary Figs. 16–17 and 7d). Different groups, including PBS, I + C, ^RM_(I+C)@CaP (without partzymes), ^RM_(I+C)@CaP (with mutated partzymes) and ^RM_(I+C)@CaP_(p) (with partzymes), were implemented under laser irradiation at 808 nm and 1 W cm⁻² for 5 min. The mutated partzyme can only bind with miRNA-21 but cannot recognize HSP70. The final concentrations in the different groups were IR780: 4 μg/ml, Cur: 2 μg/ml, and partzyme: 2 μM. After 24 h of treatment, compared with the PBS group, the pure drug I + C group showed ~20% inhibition of healthy NHDFs. However, the ^RM_(I+C)@CaP, ^RM_(I+C)@CaP_(mP) and ^RM_(I+C)@CaP_(p) groups exhibited almost no killing effect on healthy NHDFs due to less NP endocytosis and the miRNA-21 targeting effect of the MNAzyme system (Supplementary Fig. 16). For PANC-1 cancer cells, compared with the PBS group, the pure drug I + C group exhibited an ~48% inhibitory effect. However, 63% of the cancer cells were inhibited in the ^RM_(I+C)@CaP group, which suggested that Ca²⁺ enhanced the mitochondrial dysfunction effect. Approximately 75% of cells were inhibited in the ^RM_(I+C)@CaP_(mP) group (Supplementary Fig. 17), indicating that the MNAzyme system boosted cancer therapy through miRNA-21 silencing. Of note, almost 90% of the cancer cells were inhibited in the ^RM_(I+C)@CaP_(p) group, which revealed that the partzyme could achieve both miRNA and HSP70 regulation and realize an excellent synergistic effect.

From the apoptosis assay results, within 8 h of treatment (IR780: 2 μg/ml, Cur: 1 μg/ml, partzyme: 1 μM), ~13.3% of the cells in the pure drug I + C group had entered the late apoptotic phase, while the percentage was 26.0% for the ^RM_(I+C)@CaP group, reflecting Ca²⁺-mediated synergistic ion therapy. Moreover, 50.8% of the cells in the ^RM_(I+C)@CaP_(mP) group were shown to enter the late apoptotic state (Fig. 7d), which indicates the existence of a synergistic effect through mutated partzyme-based miRNA regulation. Finally, for the final formulation, 84.5% of the cells entered the late apoptotic phase, which was attributed to the MNAzyme-enhanced PTT, PTEN regulation, PDT and Ca²⁺ overload. The gating/sorting strategies are shown in Supplementary Fig. 18.

Encouraged by the selective suppressing ability and excellent biocompatibility in vitro, we further explored the performance of each nanosystem in vivo, which was approved by the Institutional Animal Care and Use Committee, Zhejiang Center of Laboratory Animals (Approval No. ZJCLA-IACUC-20020090). A pancreatic orthotopic tumor model was constructed by injecting 10⁷ PANC-1-luc cells into the pancreatic site. The blood circulation and the blood half-life of the nanomachine were investigated first to determine its pharmacokinetics and potential efficacy in vivo (Supplementary Fig. 19). Three groups, including the pure drug (IR780 + curcumin + partzymes), M_(I+C)@CaP_(p) and ^RM_(I+C)@CaP_(p) groups, were compared. From the results, we found that the M_(I+C)@CaP_(p) and ^RM_(I+C)@CaP_(p) groups had longer circulation times, with elimination half-lives (t_1/2β) of 26.69 h and 25.53 h, respectively, compared to the free drug, which had a t_1/2β of 5.34 h. The prolonged circulation time of NPs is thought to be due to the PEG shell, which shields the nanoparticle from being recognized and cleared by the immune system.

Live animal imaging was then performed to demonstrate the biodistribution of NPs after intravenous administration (Fig. 8a). Three groups, including the pure drug (IR780 + curcumin + partzymes), M_(I+C)@CaP_(p) and ^RM_(I+C)@CaP_(p) groups, were tracked at 1, 8, 24 and 48 h timepoints (IR780: 1 mg/kg). For the pure drug I + C + P group, the majority of the signal was found throughout the mouse body at the 1 h timepoint, while a negligible signal was observed after 8 h due to the rapid metabolism of IR780. In contrast, M_(I+C)@CaP_(p) NPs gradually released the drug, which reached its maximum concentration at the tumor site after 8 h of intravenous administration. Moreover, due to RGD modification, the ^RM_(I+C)@CaP_(p) group showed better tumor accumulation, achieving the highest fluorescence signal among the groups. Subsequently, the main organs were further harvested and imaged to evaluate the drug biodistribution. Compared with the free drug and M_(I+C)@CaP_(p) NPs, ^RM_(I+C)@CaP_(p) NPs resulted in pronounced fluorescence in the tumor tissue, proving that RGD can facilitate tumor targeting.

a In vivo biodistribution of nanomaterial. Tu Tumor, Li Liver, Sp Spleen, Lu Lung, Ki Kidney, He Heart. b Tumor temperature elevation pictures. c Bioluminescence at different timepoints. d Quantitative analysis of total flux signal intensity (photons/sec) over time (n = 4 independent experiments, and the data are presented as the mean values ± SDs). e Isolated tumor photos. f Tumor weight (n = 4 independent experiments, and the data are presented as the mean values ± SDs). g TUNEL labeling of each tumor slice. h TUNEL fluorescence quantitated by ImageJ (n = 3 independent experiments, and the data are presented as the mean values ± SDs). All statistics were calculated using two-tailed paired t tests, and all experiments were repeated three times independently with similar results. The source data from (d, f, h) are provided as a Source Data file.

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Later, to assess mild PTT efficiency, a tumor temperature elevation experiment was performed. Four groups (PBS, I + C + P, M_(I+C)@CaP_(p), and ^RM_(I+C)@CaP_(p)) were treated with a 1 W cm⁻² 808 nm laser at 24 h after drug administration (IR780: 1 mg/kg), and the results are elucidated in Fig. 8b. From the results, the PBS group elicited the least change in temperature even after 4 min of irradiation. The pure drug I + C + P group showed an ineffective treatment temperature after 4 min of irradiation (below 43 °C) due to the poor solubility and unstable property of IR780. The M_(I+C)@CaP_(p) NP group exhibited an enhanced photothermal conversion effect, and the tumor temperature increased to 42 °C with only 20 s of irradiation. Surprisingly, owing to the RGD-mediated enhanced tumor cell uptake, the ^RM_(I+C)@CaP_(p) group showed a dramatic temperature increase to 44.4 °C after 20 s of laser irradiation, exhibiting favorable mild photothermal activity.

Subsequently, the luciferase activity of PANC-1-luc cells within in situ carcinoma xenografts was measured to test the therapeutic efficiency of the pure drug and ^RM_(I+C)@CaP nanosystems with/without partzymes in vivo (Fig. 8c). The quantitative analysis of luciferase intensity is shown in Fig. 8d. First, the results showed that the pure drug with laser group (I + C + P + Laser) could not effectively inhibit tumor growth, which might have been due to the easy degradation of the pure drug in vivo. In contrast, the ^RM_(I+C)@CaP +Laser group effectively inhibited tumor growth for the first 10 days during the treatment period due to its excellent photothermal therapeutic effect and tumor targeting. However, the tumor showed a second round of growth after the 20th day, and the total flux changed from 5.09 × 10⁸ p/s to ~2.72 × 10⁹ p/s, which might be attributable to acquired drug and heat resistance. Surprisingly, when partzyme was encapsulated into the nanosystems, the therapeutic efficacy of the ^RM_(I+C)@CaP_(P) +Laser group increased significantly and continued until Day 40, and the total flux equaled 5.04 × 10⁸ p/s. It is possible that after the HSP70 gene was silenced by the MNAzyme, the tumor was unable to produce heat resistance, and the silencing of miRNA-21 also promoted the expression of the tumor suppressor gene PTEN protein and accelerated the apoptosis of the tumor. Furthermore, the photographs of tumors in situ (Fig. 8e) and tumor weight (Fig. 8f) indicated that the ^RM_(I+C)@CaP_(P) +Laser group had the best therapeutic effect, and its tumor weight was 0.049 g, which was ~50% of that of the PBS group. The TUNEL assay also demonstrated the therapeutic efficiency of partzyme (Fig. 8g), and the ^RM_(I+C)@CaP_(P) +Laser group revealed an 18% greater TUNEL signal than the ^RM_(I+C)@CaP without partzymes +Laser group (Fig. 8h).

As mentioned previously, HSP70 can protect healthy cells from high temperature, but it is also the resistance mechanism for cancer cells against photothermal therapy (Fig. 9a). Therefore, smart nanomachines that can process biological signal input (miRNA-21) will significantly increase the selectivity and efficiency of PTT (Fig. 9b). To further verify the tumor-targeted silencing function of our nanodevices, the gene and protein expression levels of HSP70 were evaluated in both tumor and paracancerous tissues. Pure drug and ^RM_(I+C)@CaP nanosystem with/without partzymes groups were used.

a Roles of HSPs in different cells. b Tumor-targeting mechanism for our intelligent nanodevice. c Immunohistochemical characterization of HSP70 in tumor tissues and healthy tissues. d HSP70 quantitated by ImageJ in tumor tissue and paracancerous tissue (n = 3 independent experiments, and the data are presented as the mean values ± SDs). e qPCR results for HSP70 mRNA levels (n = 3 independent experiments, and the data are presented as the mean values ± SDs). f, g miRNA-21 content and PTEN protein expression levels (n = 3 independent experiments). All statistics were calculated using two-tailed paired t tests, and all experiments (c, f, g) were repeated three times independently with similar results. The source data from (d, e) are provided as a Source Data file.

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For tumor tissue, the HSP70 protein expression level in the pure drug group increased by ~66% under laser irradiation (Fig. 9c, d) compared with that in the PBS +Laser group. Moreover, the ^RM_(I+C)@CaP (without partzymes) +Laser group also showed ~34% HSP70 upregulation. Previous in vitro experiments showed that ^RM_(I+C)@CaP could downregulate HSP70 by regulating mitochondrial function (Fig. 9e). However, these in vivo experiments suggested that the nanomachine could mediate more efficient photothermal conversion efficiency than the pure drug, which exacerbated the production of HSP70 (Fig. 9c, d). Hence, the enhancement of HSP70 expression could explain how the PTT resistance caused a second round of tumor growth (Fig. 9d). Importantly, the nanocarrier loaded with partzymes (^RM_(I+C)@CaP_(P)) could achieve effective downregulation of HSP70 even under laser irradiation (Fig. 9c, d), which proved the successful MNAzyme assembly. The results of the western blot experiment also provided ample evidence of the silencing effect of MNAzymes on HSP70 (Supplementary Fig. 20).

Nevertheless, in healthy pancreatic tissue, the HSP70 levels at the outer edge of healthy tissue were elevated in the pure drug and ^RM_(I+C)@CaP groups as well as in the ^RM_(I+C)@CaP_(P) +Laser group. Hence, as we expected, NPs and drugs inevitably penetrated healthy paracancerous tissues, but our intelligent nanomachine was able to distinguish cancer cells from healthy cells by virtue of its tumor signal recognition function. Later, the qPCR results correlated with the above phenomenon. Compared with those in the ^RM_(I+C)@CaP group, the HSP70 mRNA levels within the ^RM_(I+C)@CaP_(P) group were reduced by nearly 90% (Fig. 9e) in tumor tissue. However, for healthy tissues around tumors, our intelligent nanosystem did not affect HSP70-based self-protection, thereby remarkably improving the targeting of the treatment.

Subsequently, the MNAzyme system-mediated miR-21 silencing effect was further studied. The miR-21 content was measured through fluorescence in situ hybridization (FISH) (Fig. 9f), and the downstream protein PTEN was characterized through IHC (Fig. 9g). First, the results clearly showed that the content of miR-21 had no relationship with laser irradiation and that the control group contained the highest amount of miR-21. The content of miR-21 decreased in both the pure drug and ^RM_(I+C)@CaP groups since Cur can reduce the level of miR-21. However, we found that miR-21 was almost completely silenced within the partzyme-containing ^RM_(I+C)@CaP_(P) group. These results indicated that partzymes can utilize and consume miRNA at tumor sites, which can further achieve miR-21 silencing-based gene therapy (Fig. 9g).

PTEN has been found to be a tumor suppressor with growth and survival regulatory functions, and silencing of miR-21 can mediate PTEN upregulation, which in turn inhibits tumor growth⁶⁵. The results in Fig. 9g revealed that, for the ^RM_(I+C)@CaP and I + C + P groups, the PTEN levels were all increased, which was ascribed to Cur. The ^RM_(I+C)@CaP_(P) group exhibited the highest PTEN level due to the excellent miR-21 silencing effects of both Cur and the MNAzyme system. These results reflect the cleverness of our designed MNAzyme. It can not only recognize miR-21 and use it as the building block for MNAzyme self-assembly but also silence miR-21 to achieve gene therapy.

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• Source: https://www.nature.com/articles/s41467-023-42740-2