Abstract

   Effective pyroptosis induction is a promising approach to potentiate
   cancer immunotherapy. However, the actual efficacy of the present
   pyroptosis inducers can be weakened by successive biological barriers.
   Here, a cascaded pH‐activated supramolecular nanoprodrug (PDNP) with a
   stepwise size shrinkage property is developed as a pyroptosis inducer
   to boost antitumor immune response. PDNPs comprise multiple
   poly(ethylene glycol) (PEG) and doxorubicin (DOX) drug–polymer hybrid
   repeating blocks conjugated by ultra‐pH‐sensitive benzoic imine (bzi)
   and hydrazone (hyd) bonds. The PEG units endow its “stealth” property
   and ensure sufficient tumor accumulation. A sharp switch in particle
   size and detachment of PEG shielding can be triggered by the acidic
   extracellular pH to achieve deep intratumor penetration. Following
   endocytosis, second‐stage size switching can be initiated by more
   acidic endolysosomes, and PDNPs disassociate into ultrasmall cargo to
   ensure accurate intracellular delivery. The cascaded pH activation of
   PDNPs can effectively elicit gasdermin E (GSDME)‐mediated pyroptosis to
   enhance the immunological response. In combination with anti‐PD‐1
   antibody, PDNPs can amplify tumor suppression and extend the survival
   of mice, which suggests a powerful immune adjuvant and pave the way for
   high‐efficiency immune checkpoint blockade therapy.

   Keywords: cancer immunotherapy, pH cascaded responsiveness, pyroptosis,
   size conversion, supramolecular prodrug
     __________________________________________________________________

   A cascaded pH‐activated supramolecular nanoprodrug (PDNP) with
   mutistage size shrinkage property is developed to combat the successive
   drug delivery barriers. The size‐transformable PDNPs can precise
   intracellular delivery drug for effectively eliciting pyroptosis and
   augmenting antitumor immune response, favorable for boosting checkpoint
   blockade‐based immunotherapy.

   graphic file with name ADVS-9-2203353-g007.jpg

1. Introduction

   The immune system has a high degree of plasticity, which significantly
   affects tumor progression.^[ [40]^1 ^] It is constantly regulated by
   immune elimination, equilibrium, and escape in the tumor
   microenvironment (TME).^[ [41]^2 ^] Checkpoint blockade‐based
   immunotherapy has emerged as a promising clinical approach for
   malignancies that can effectively alleviate immune evasion by impeding
   the binding of programmed death receptors and their ligands, thereby
   increasing the aggressiveness of the host immune system against tumor
   cells.^[ [42]^3 ^] However, its practical efficacy has long been
   limited by inferior activation of the immune response.^[ [43]^4 ^]
   Accumulating evidence suggests that tumors possessing high
   immunogenicity are pivotal for modulating the immunosuppression and
   boosting the immune response, which directly affects the activation and
   infiltration of cytotoxic T lymphocytes (CTLs).^[ [44]^5 ^] Recent
   findings on pyroptosis have elucidated its association with adaptive
   immunity, providing new insights for tumor immunotherapy.^[ [45]^6 ^]
   Pyroptosis is an inflammatory form of gasdermin (GSDM)‐mediated
   programmed cell death induced by various stimuli, such as viral,
   bacterial, and chemotherapy drugs.^[ [46]^7 ^] Cleavage of the GSDM
   protein family by active caspase releases the GSDM‐N fragments that
   punch holes in the cytomembrane, resulting in morphological variation
   involving membrane blebbing, cell rupture, and the leakage of
   cytokines, which can directly activate the immune system and positively
   regulate the immune response.^[ [47]^8 ^] A variety of chemotherapeutic
   drugs, such as doxorubicin (DOX), have shown to induce
   caspase‐3‐mediated pyroptosis in tumor cells with high gasdermin E
   (GSDME) expression, which potentiates the immunogenicity of tumors and
   elicits robust antitumor immunity.^[ [48]^9 ^] Despite the potential of
   pyroptosis for the enhancement of immunotherapy, the actual efficacy of
   the present pyroptosis inducers can be weakened by the successive
   biological barriers during drug delivery, resulting in failure to
   achieve the expected efficiency and causing severe adverse effects.^[
   [49]^10 ^]

   Current nanodelivery systems can partially address these issues.^[
   [50]^11 ^] For example, clinically approved PEGylated liposomal
   doxorubicin (Doxil) effectively enhanced tumor accumulation and
   relieved adverse effects.^[ [51]^12 ^] However, the therapeutic
   outcomes show no considerable improvements compared to monotherapy.^[
   [52]^13 ^] Most nanomedicines fail to overcome the successive restrains
   of complicated delivery journeys, because there are different
   requirements for the properties of nanomedicines at different delivery
   phases.^[ [53]^14 ^] Theoretically, there is a diminishing “size
   ladder” principle for nanomedicines to be transported from the blood to
   tumor nucleus.^[ [54]^15 ^] Larger nanoparticles (≈100 nm) tend to
   accumulate at tumor sites via the enhanced permeability and retention
   effect, while the dense extracellular matrix, interstitial fluid
   pressure, and a high degree of hypovascularity hamper their deep
   penetration and retention in the perivascular regions of solid
   tumors.^[ [55]^16 ^] It has been reported that nanoparticles (≈30 nm)
   can alleviate diffusional resistance in the tumor interstitial space
   and facilitate intratumor penetration, whereas they are easily
   eliminated during blood circulation due to the poor retention effect.^[
   [56]^15 , [57]^17 ^] Moreover, it requires the nanoparticles with
   smaller size to accurately enter the nucleus due to the nuclear pore
   threshold (<9 nm).^[ [58]^18 ^] Although most nanomedicines can
   surmount some of these restraints, any one of the unconquered obstacles
   can be an Achilles’ heel lessens the final drug amounts inside the
   tumor cells.

   Herein, we present a drug–polymer hybrid supramolecular nanoprodrug
   (PDNP) as a pyroptosis inducer to potentiate tumor immunogenicity for a
   robust antitumor immune response (Scheme [59] 1 ). PDNPs are fabricated
   by multiple drug–polymer repeating units, which are comprised of
   PEGylated DOX blocks, integrated by two different acid‐cleavable
   linkers to respond to the increasing acidity of the TME. At
   physiological pH, the PDNPs retain the “stealth” property of PEG to
   ensure sufficient tumor accumulation. Upon arrival at the acidic TME
   (pH ≈ 6.5), PDNPs begin to detach from PEG shielding and rapidly
   dissociate into small particles because the protonation of the
   ultra‐pH‐sensitive benzoic imine bond for deep tumor penetration. After
   endocytosis, second‐stage size shrinkage occurs because the cleavage of
   the hydrazone bond in response to the more acidic endolysosome (pH ≈
   5.0), resulting in complete decomposition of PDNPs and release of DOX
   into the nucleus. Effective penetration and accurate release of drugs
   in the tumor can effectively provoke GSDME‐mediated pyroptosis, which
   facilitates dendritic cell (DC) maturation, prime CTL proliferation,
   and inhibiting myeloid‐derived suppressor cells (MDSCs), thereby
   boosting the antitumor immune response. Owing to their
   pyroptosis‐induced immune‐potentiating effects, PDNPs can sensitize
   tumors to anti‐PD‐1 checkpoint blockade therapy, amplify therapeutic
   efficacy, and prolong survival; in cooperation with immune checkpoint
   inhibitors, they may be a powerful clinical candidate for boosting
   cancer chemo‐immunotherapy.

Scheme 1.

   Scheme 1
   [60]Open in a new tab

   Schematic illustration of size‐transformable supramolecular nanoprodrug
   (PDNP)‐mediated cancer chemo‐immunotherapy. A) Chemical structure and
   fabrication of PDNPs and the mechanism of pH cascade‐responsive
   conversion and drug release. B) Illustration of PDNP‐induced pyroptosis
   and positive regulation of antitumor immunity.

2. Results and Discussion

   PDNPs were synthesized by a two‐kind aminaldehyde reaction of
   aldehylated PEG blocks and hydrazone bond‐modified DOX prodrugs (Figure
   [61]S1, Supporting Information), forming a drug–polymer hybrid
   supramolecular prodrug (DOX‐bzi‐PEG‐hyd‐DOX). The chemical structures
   were confirmed using ^1H NMR spectroscopy and an FTIR spectrophotometer
   (Figures [62]S2–S5, Supporting Information). The characteristic peak of
   DOX in the fluorescence spectrum and ultraviolet (UV) absorption was
   observed in the PDNPs, confirming the successful integration of DOX
   (Figure [63]S6, Supporting Information). The number‐average molecular
   weight (M[n]) and weight‐average molecular weight (M[w]) of PDNPs were
   1.2 × 10^4 and 2.6 × 10^4, respectively, demonstrating that PDNPs were
   integrated by multiple repeated drug–polymer blocks (Figure [64]S7,
   Supporting Information). The unique structure of PDNPs endows it with a
   high drug loading capacity of 12.6 ± 0.7 wt%, as determined by UV–vis
   absorbance (Figure [65]S8, Supporting Information). The PDNPs showed a
   negative surface charge of −14.6 ± 1.4 mV (Figure [66]S9, Supporting
   Information). The hemolysis rate of PDNPs was lower than 5% in the
   range of 1–200 µg mL^−1, indicating their superior blood compatibility
   (Figure [67]S10, Supporting Information). The stability of PDNPs was
   examined by dynamic light scattering (DLS) measurements over a period
   of 7 days in PBS, and there were slight but negligible changes in size
   and zeta potential (Figure [68] 1E). Furthermore, PDNPs maintained good
   stability and polydispersity in DMEM or PBS with 10% FBS at 48 h
   (Figure [69]S11, Supporting Information), suggesting that PDNPs possess
   desirable stability during blood circulation, thus ensuring effective
   tumor accumulation.

Figure 1.

   Figure 1
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   Characterization and working mechanism of PDNPs. A) Schematic
   illustration of pH‐triggered sequential conversion of PDNPs. B) TEM
   images of PDNPs at pH 7.4, 6.5, and 5.0. C) Particle size distribution
   of PDNPs at different pH values over time. D) The drug release profiles
   of PDNPs in PBS solutions with different pH values (data presented as
   mean ± SD, n = 3). E) The stability of PDNPs within 7 days (data
   presented as mean ± SD, n = 3). F) Cellular uptake count (histogram)
   and the mean fluorescence intensity (M.F.I) (scattergram) of PDNPs in
   CT26 cells at pH 7.4 or 6.5 (data presented as mean ± SD, n = 3,
   P‐values are calculated using Student's t‐test, *P < 0.05, ***P <
   0.001). G) Cell viability at pH 7.4 or 6.5 (data presented as mean ±
   SD, n = 3). H) Acridine orange (AO) staining to indicate lysosomal
   disruption in CT26 cells treated with PDNPs. I,J) The M.F.I and CLSM
   images of DNA damage in CT26 cells incubated with PDNPs and free DOX
   (data presented as mean ± SD, n = 3, P‐values are calculated using
   one‐way ANOVA, *P < 0.05, ***P < 0.001). K) TEM images of CT26 cell
   treated with PDNPs at different time points.

   To test cascaded acid sensitivity, PDNPs were incubated in solutions
   with different pH values. The mechanism of pH‐triggered sequential
   conversion is illustrated in Figure [71]1A, and its morphology and size
   were measured using transmission electron microscopy (TEM) and DLS. At
   pH 7.4, the PDNPs showed a spherical shape with an average size of
   129.7 ± 8.2 nm. PDNPs shrunk to 37.9 ± 5.9 nm at pH 6.5 and finally
   completely decomposed into 8.1 ± 3.2 nm at pH 5.0 (Figure [72]1B),
   which was consistent with the DLS results (Figure [73]S12, Supporting
   Information). The variations in the particle size of PDNPs incubated in
   different pH solutions over time are shown in Figure [74]1C, confirming
   the stepwise size shrinkage property of PDNPs owing to the cleavage of
   acid‐labile linkage and PEG detachment.

   To investigate the pH‐triggered drug release behavior of PDNPs,
   cumulative DOX release from PDNPs was evaluated in PBS at pH 7.4, 6.5,
   and 5.0. The release profile of the PDNPs showed a sustained release of
   DOX over 72 h under diverse conditions. There was a rapid drug release
   of 43.4% and 86.6% at pH 6.5 and 5.0, respectively; however, only 18.6%
   DOX was released from PDNPs at pH 7.4 (Figure [75]1D). PDNPs showed an
   increase in cumulative drug release at pH 6.5 compared with that at pH
   7.4, which was ascribed to the protonation of the benzoic imine moiety
   in PDNPs in a weakly acidic environment. The drug release rate of PDNPs
   at pH 5.0 was faster than that at pH 6.5 because of the cleavage of the
   hydrazone bond at more acidic pH conditions. These results were
   consistent with the fluorescence spectrometer, which showed a
   significant increase in fluorescence intensity of ≈2.2‐fold and
   ≈2.8‐fold at pH 6.5 and 5.0, respectively, compared to that at pH 7.4,
   confirming that the sequential dissociation of PDNPs was triggered at
   different pH ranges (Figure [76]S13, Supporting Information).

   Although PEGylation has been proven to be an effective strategy for
   prolonging circulation, the resulting inefficiency of cellular uptake
   remains a concern.^[ [77]^19 ^] By virtue of the introduction of an
   acid‐labile linkage, dePEGylation can be triggered in response to an
   acidic TME, which is capable of amplifying the intracellular delivery
   efficiency by PEG deshielding. Therefore, we investigated the
   acid‐dependent intracellular activation of PDNPs in murine colon cancer
   (CT26) cell lines. A strong fluorescence signal of DOX was observed in
   CT26 cells after 4 h of coincubation with PDNPs at pH 7.4, while there
   was an obvious signal augmentation with the pH value decreasing to 6.5
   (Figure [78]S14, Supporting Information). Flow cytometry was used to
   further quantify the amount of DOX internalized by the PDNPs (Figure
   [79]S15, Supporting Information). At pH 6.5, the percentage of positive
   cells incubated with PDNPs after 4 h was ≈1.2‐fold higher than that at
   pH 7.4. The mean fluorescence intensity of the positive cells also
   exhibited a significant elevation at pH 6.5 (Figure [80]1F), confirming
   the enhancement of cellular uptake attribute to acid‐triggered PEG
   detachment. The cytotoxicity of PDNPs was evaluated by MTT and
   live‐staining assays in CT26 cell lines (Figure [81]1G; Figure [82]S16,
   Supporting Information). PDNPs exhibited dose‐dependent antitumor
   efficacy, and cell viability was dramatically reduced upon incubation
   with PDNPs at pH 6.5 than that at pH 7.4. Moreover, the half‐inhibitory
   concentration (IC[50]) value of the PDNPs in CT26 cells at pH 7.4 was
   6.8 μg/mL, whereas it was 3.2 μg/mL at pH 6.5 (Figure [83]S17,
   Supporting Information). The increased cytotoxicity of PDNPs at pH 6.5
   could be ascribed to more efficient cellular uptake and higher DOX
   release under acidic conditions.

   To explore the intracellular trafficking pathway and working mechanism
   of PDNPs, we first evaluated the colocalization efficiency of PDNPs
   with lysosomes. As shown in Figure [84]S18 of the Supporting
   Information, the overlapping fluorescence signal between PDNPs and
   endolysosomes first increased and then decreased over time, indicating
   that PDNPs could effectively target and escape from lysosomes. The
   Pearson correlation coefficient quantitatively affirmed the
   time‐determined escape of PDNPs from lysosomes, which was ascribed to
   the ionization of PDNPs in the acidic compartment, followed by the “pH
   sponge effect” giving rise to lysosomal damage. We then evaluated the
   disruption of lysosomes by PDNPs using acridine orange staining. As
   shown in Figure [85]1H, the red signals assigned to lysosomes almost
   disappeared after 12 h of incubation with PDNPs, and the green
   fluorescence reflected a shrinking morphology due to cell apoptosis. As
   a genotoxic therapeutic agent, DOX induces apoptosis by interfering
   with topoisomerase II‐mediated DNA replication and transcription. Thus,
   we detected the degree of DNA damage caused by the PDNPs using γ‐H2AX
   antibody immunofluorescence staining (Figure [86]1I,J). Vast
   accumulation of green fluorescence was observed in the nucleus after
   PDNPs treatment for 24 h, indicating that PDNPs could elicit intense
   DNA damage. Furthermore, we observed the intracellular trafficking
   pathway of PDNPs in CT26 cells using TEM. As shown in Figure [87]1K, a
   mass of small nanoparticles with a size of ≈10 nm rapidly accumulated
   in lysosomes after 4 h of incubation, confirming the endocytosis
   mechanism of PDNPs in acidic endolysosomes, with a size‐transformable
   performance. After 8 h, the lysosomes were disrupted and the
   nanoparticles were prone to escape from them into the cytoplasm. After
   incubation for 24 h, many tiny particles covered the entire cytoplasm
   and tended to accumulate in the nucleus, leading to apoptosis. Taken
   together, these results implied that PDNPs could dissociate into small
   nanoparticles in response to the acidic endolysosomes and escape from
   the endolysosomal chamber due to the proton sponge effect, avoiding
   elimination by the exocytosis pathway without drug release,
   facilitating active drug molecule access to the nucleus, and inducing
   cell apoptosis.

   Effective antigen presentation is an indispensable prerequisite for T
   cell activation. Recent studies have asserted that DOX induces
   pyroptosis by activating caspase‐3 to cleave GSDME.^[ [88]^9a ^] The
   resulting N‐terminal domains of GSDME would elicit membrane perforation
   and cell burst, accompanied by secretion of high‐mobility group box 1
   (HMGB1) and release of lactate dehydrogenase (LDH) and adenosine
   triphosphate (ATP) to promote DCs maturation and antigen presentation
   to enhance the immunological response (Figure [89] 2B). Thus, we
   speculated that PDNPs could effectively induce pyroptosis to stimulate
   the immune response. First, we examined whether CT26 cells exhibit the
   morphological characteristics of pyroptosis upon PDNPs treatment. As
   shown in Figure [90]2A, features of pyroptosis with cell swelling and
   ballooning from the plasma membrane were observed after 6 h of
   treatment with PDNPs. At 12 h, pyroptosis occurred in most cells and
   the cells tended to burst after 24 h of treatment with PDNPs.
   Considering that PEGylated liposomal doxorubicin (Lipo‐Dox) with a
   particle size of ≈180 nm is an FDA approved clinical preparation, we
   used it for comparison (Figure [91]S19, Supporting Information). As
   shown in Figure [92]2E, the images of cells with different treatments
   displayed an increased number of pyroptotic cells. Next, we determined
   the release of ATP and LDH in CT26 cell line, and the high release
   content of ATP and LDH was measured in the supernatant of PDNP‐treated
   cells (Figure [93]2C,D).

Figure 2.

   Figure 2
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   Pyroptosis and immunostimulatory behaviors of PDNPs. A) Representative
   bright‐field CLSM images of CT26 cells treated with PDNPs at different
   time period. Arrows denote the pyroptotic cells. B) Schematic
   illustration of PDNPs‐induced pyroptosis mechanism. C,D) The release of
   ATP and LDH from CT26 cells after different treatments (Data presented
   as mean ± SD, n = 3, P‐values are calculated using one‐way ANOVA, *P <
   0.05, **P < 0.01, ***P < 0.001). E) Representative bright‐field CLSM
   images of CT26 cells with different treatments. F) Western blotting
   detection of full‐length GSDME (GSDME‐FL), GSDME‐N terminal, and
   Cleaved caspase‐3 (CL‐caspase‐3) in CT26 cells and tumor tissues upon
   PDNPs treatment (data presented as mean ± SD, n = 3). G,H) The relative
   GSDME‐N expression levels and CL‐caspase‐3 expression levels in CT26
   tumor tissues (data presented as mean ± SD, n = 3, P‐values are
   calculated using one‐way ANOVA with Bonferroni correction, *P < 0.05).
   I) The representative CLSM images of HMGB1 and CRT in CT26 cells. J)
   The representative immunohistochemical staining of granzyme B and
   CL‐caspase‐3 in tumor tissues upon different treatments.

   We next detected pyroptosis‐related protein expression in vitro and in
   vivo using western blotting. As shown in Figure [95]2F–H and Figure
   [96]S20 (Supporting Information), high‐level expression of GSDME‐N and
   cleaved caspase‐3 (CL‐caspase‐3) was observed in PDNP‐treated CT26 cell
   lines and tumor tissues, confirming that active caspase‐3 effectively
   cleaved the linkage of GSDME to generate the GSDME‐N domain and induce
   pyroptosis. Similar results were observed in murine breast cancer cell
   lines (4T1) (Figure [97]S21, Supporting Information). Thereafter, cell
   membrane exposure of calreticulin (CRT) and extracellular release of
   HMGB1 were evaluated to verify the enhancement of PDNP‐induced
   immunogenicity. Figure [98]2I shows obvious HMGB1 release and CRT
   exposure in PDNP‐treated cells. Quantitative analysis showed that PDNPs
   significantly elevated HMGB1 release and CRT exposure, which were ≈2.7‐
   and ≈6.4‐fold higher than those in the control group (Figure [99]S22,
   Supporting Information). In addition, granzyme B can directly shear
   GSDME and activate pyroptosis in tumor cells. Thus, we examined the
   expression of granzyme B and CL‐caspase‐3 in the tumor tissues by
   immunohistochemistry, and the local increase was clearly observed in
   the PDNP‐treated group (Figure [100]2J; Figure [101]S23, Supporting
   Information).

   To further understand the mechanisms of pyroptosis and related immune
   responses induced by PDNPs, we evaluated gene expression by RNA‐seq in
   CT26 cells after treatments, and the principal component analysis, as
   shown in Figure [102]S24 of the Supporting Information. As depicted in
   the volcano plot (Figure [103] 3A), 6941 differential genes were
   detected in the PDNP‐treated groups compared with the control group
   (|log[2] fold change| > 1 and adjusted P‐value < 0.05), including 3142
   upregulated genes and 3799 downregulated genes. The Venn diagram shows
   all the expressed genes in the control and PDNPs groups
   (Figure [104]3B). We then analyzed the mRNA expression levels of genes
   associated with pyroptosis and the immune system and found that genes
   related to inflammatory cytokines, antigen processing and presentation,
   and T cell functional molecules were distinctly upregulated in the
   PDNP‐treated group (Figure [105]3D). As shown in Figure [106]3C, these
   upregulated genes were closely associated with each other and
   participated individually or jointly in the innate and adaptive immune
   responses. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
   enrichment analysis indicated that after PDNPs treatment,
   differentially expressed genes were enriched in inflammatory‐related
   signaling pathways, apoptosis‐mediated signaling pathways, and immune
   response‐associated signaling pathways, such as TNF signaling pathways,
   NOD‐like receptor signaling pathway, MAPK signaling pathway, RIG‐I‐like
   receptor signaling pathway, and Toll‐like receptor signaling pathway
   (Figure [107]3E). Gene ontology (GO) enrichment analysis further
   revealed a greater rich ratio upon treatment with PDNPs being mainly
   involved in inflammatory responses, regulation of response to cytokine
   stimulus, innate immune response, and T cell differentiation
   (Figure [108]3F). These results confirm that PDNPs treatment
   efficiently elicit pyroptosis to potentiate tumor immunogenicity for a
   robust antitumor immune response.

Figure 3.

   Figure 3
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   RNA sequencing analysis. A) Volcano plot of the distributions of
   differentially expressed genes after PDNPs treatment (|log[2] fold
   change| > 1, adjusted P‐value < 0.05). B) Venn diagram of all expressed
   genes in each group. C) Functional associated networks of the
   differentially expressed genes from the STRING database. D) Heatmap
   analysis of mRNA expression levels of genes involved in pyroptosis and
   immune system. E) KEGG pathway analysis and F) GO enrichment after
   PDNPs treatment. Data presented as mean ± SD, n = 3.

   The potency of sufficient tumor accumulation and deep intratumor
   penetration seems contradictory for nanomedicine. Nanoparticles with a
   small size have been proven to be favorable for diffusion into tumors,
   but are easily cleared by the blood circulation system. Since PDNPs
   possess size‐transformable properties in response to the acidic tumor
   extracellular pH, they were expected to show enhanced intratumor
   infiltration and simultaneously retain the capacity of long blood
   circulation. To confirm this, 3D tumor models fabricated by CT26
   multicellular spheroids (MCSs) were utilized to evaluate the
   penetration capability of PDNPs in vitro and the schematic illustration
   is shown in Figure [110] 4B. After incubation with PDNPs in pH 7.4 and
   6.5 medium for 6 h, the red fluorescence of PDNPs perfused throughout
   the MCSs at pH 6.5, in contrast with the limited distribution around
   the MCSs periphery at pH 7.4 (Figure [111]4A; Figure [112]S25,
   Supporting Information), demonstrating that the acid‐triggered size
   conversion was a feasible strategy to facilitate particle traversing of
   the dense tumor matrix. The pharmacokinetics of PDNPs were then studied
   by detecting the plasma levels of DOX after intravenous injection in
   S.D. rats. The circulating half‐life (t [1/2]) and AUC value of PDNPs
   were ≈3.7‐fold and ≈2.9‐fold higher than those of free DOX,
   respectively (Figure [113]4C,D; Figure [114]S26, Supporting
   Information), and the pharmacokinetics parameters were presented in
   Table [115]S1 of the Supporting Information, indicating the preferable
   retention time of PDNPs in vivo.

Figure 4.

   Figure 4
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   Penetration, pharmacokinetics, and biodistribution studies of PDNPs. A)
   The representative CLSM images of CT26 MCSs at varied depths upon
   incubation with PDNPs for 6 h at pH 7.4 or 6.5 (data presented as mean
   ± SD, n = 3). B) Schematic illustration of the acid‐triggered
   size‐transformable PDNPs for enhanced intratumor penetration in CT26
   MCSs. C) Plasma concentration of DOX versus time of PDNPs upon
   administration of PDNPs and free DOX in S.D. rats (data presented as
   mean ± SD, n = 3). D) The circulating half‐life (t [1/2]) of PDNPs and
   free DOX in S.D. rats (data presented as mean ± SD, n = 3, P‐values are
   calculated using Student's t‐test, **P < 0.01). E) The representative
   in vivo NIR imaging of free DiR and PDNPs@DiR in CT26‐bearing mice. Red
   circles denote the tumor region (data presented as mean ± SD, n = 3).
   F) The representative CLSM images of the distribution and penetration
   of PDNPs in the slices of CT26 tumor tissue. G) Quantitative analysis
   of the excised tumors and organs at 48 h postinjection (data presented
   as mean ± SD, n = 3, P‐values are calculated using Student's t‐test,
   ***P < 0.001). H) The representative immunofluorescence staining of
   excised tumor tissues after treatments for penetration studies. (DAPI:
   blue; DOX: red; blood vessels: green, stained with CD31 antibody.)

   Thereafter, the in vivo tumor accumulation and distribution of PDNPs
   were investigated after their administration into CT26 tumor‐bearing
   mice. We first loaded the lipophilic near‐infrared (NIR) fluorescent
   dye DiR into the PDNPs (denoted PDNPs@DiR). The in vivo NIR
   fluorescence images showed that the PDNPs@DiR‐treated group showed more
   intense fluorescence than those of free DiR treated group at 6, 12, 24,
   and 48 h postinjection in the tumor region (Figure [117]4E; Figures
   [118]S27 and [119]S28, Supporting Information). After 48 h, the tumor
   fluorescence in the PDNPs@DiR treated group was still strong, and the
   fluorescence intensity was ≈6.1‐fold higher than that of the free
   DiR‐treated group (Figure [120]4G). We then examined the tumor sections
   using CLSM. As shown in Figure [121]4F, extensive signals from the
   PDNPs@DiR group were observed in the tumor parenchyma. By contrast, the
   signals of the free DiR group were confined to the peripheral tumor,
   and it was difficult to infiltrate deeply into the tumors. The
   corresponding quantitative analysis of the DiR distribution is shown in
   Figure [122]S29 of the Supporting Information. We further investigated
   whether PDNPs could effectively extravasate from tumor vessels by
   examining intratumor distribution with immunofluorescence staining.
   After in vivo experiments, the harvested tumors were stained with
   anti‐CD31 antibodies that indicated tumor vascular endothelium. As
   shown in Figure [123]4H and Figure [124]S30 (Supporting Information),
   PDNPs could highly diffuse from the tumor vessels and have a more
   extensive fluorescence distribution. By contrast, the signals of
   Lipo‐Dox were almost confined to the perivascular area of the tumor,
   and it was difficult to penetrate into the deep regions, which might be
   ascribed to the limitation of particle size. These results indicate
   that size‐transformable PDNPs could address the need for both long
   blood circulation and deep intratumor penetration.

   Next, the in vivo antitumor efficiency of PDNPs was evaluated in BALB/c
   mice bearing CT26 or 4T1 tumor xenografts (Figure [125]5A–D). All
   animal experiments were conducted in accordance with the animal
   research guidelines approved by the Experimental Animal Ethics
   Committee of the School and Hospital of Stomatology, Wuhan University
   (S07920080J). Tumor volume increased rapidly in the saline‐treated
   group. Lipo‐Dox and free DOX treatments resulted in moderate
   therapeutic effects, with ≈43% and ≈49% (CT26 tumor model) or ≈37% and
   ≈35% (4T1 tumor model) inhibition rates, respectively, versus saline
   treatment. By contrast, PDNP treatments resulted in the highest
   antitumor activity, with ≈74% (CT26 tumor model) or ≈85% (4T1 tumor
   model) tumor suppression (Figures [126]S31 and [127]S32, Supporting
   Information). Hematoxylin and eosin staining and TUNEL staining in both
   tumor models showed evident tumor necrosis and increased cell apoptosis
   induced by PDNPs. Ki67 staining revealed the lowest level of
   proliferating cells after treatment with PDNPs (Figures [128]S33 and
   [129]S34, Supporting Information). Furthermore, no significant weight
   loss, spleen inflammation, or cardiac tissue damage were observed after
   PDNPs treatment (Figures [130]S35–S40, Supporting Information).
   Hematological assays showed no obvious abnormalities, indicating that
   the PDNPs were of prominent biosafety (Figure [131]S41, Supporting
   Information). These results demonstrate that the size‐transformable
   PDNPs have prolonged circulation time, enhanced tumor accumulation,
   deep intratumor penetration, effective cellular uptake, and sufficient
   drug release, thereby resulting in superior therapeutic outcomes and
   reduced adverse effects.

Figure 5.

   Figure 5
   [132]Open in a new tab

   In vivo antitumor activity and enhanced immune responses for combined
   anti‐PD‐1 therapy. A–D) Therapeutic schedule for tumor treatments in
   CT26 or 4T1‐bearing mice models and the corresponding growth curves
   upon different treatments. E,F) Flow cytometric analysis and
   quantitation of CD80^+ CD86^+ DCs on CD11c cells in spleen (SP). G,H)
   Flow cytometric analysis and quantitation of CD44^+ CD62L^+ central
   memory T (T[CM]) cells on CD8^+ cells in SP. I,J) Flow cytometric
   analyses and quantitation of Ly6C^high Ly6G^− M‐MDSCs on CD11b cells in
   SP. K,L) The representative immunofluorescence and immunohistochemical
   analysis of DC maturation and T cell infiltration in the tumor. M)
   Therapeutic strategy of PDNPs combined with anti‐PD‐1 therapy in
   CT26‐bearing mice (red arrows denote PDNPs administration; black arrows
   denote anti‐PD‐1 administration). N) Growth curves of the combination
   therapy upon different treatments in CT26‐bearing mice. O) Schematic
   illustration of the mechanism of PDNPs‐induced pyroptosis for effective
   anti‐PD‐1 checkpoint blockade therapy. P) Survival curves of the
   combination therapy upon different treatments. Q) Bioluminescence
   imaging of CT26‐bearing mice during the experiment. Data presented as
   mean ± SD, n = 5, P‐values are calculated using one‐way ANOVA, *P <
   0.05, **P < 0.01, ***P < 0.001.

   Having demonstrated the immune‐potentiating effects of PDNPs by
   inducing pyroptosis, we further explored whether PDNPs could evoke
   immune responses by harvesting spleens and tumors from CT26
   tumor‐bearing mice after treatment using flow cytometry,
   immunofluorescence, and immunohistochemical analysis. The gating
   strategies used in flow cytometry analysis are shown in Figures
   [133]S42–S44 of the Supporting Information. The results revealed that
   the percentage of mature CD80^+ CD86^+ DCs and CD44^+ CD62L^+ central
   memory T cells (T[CM]) within the spleen were obviously upregulated
   after PDNPs treatment for immune response (Figure [134]5E–H).
   Furthermore, MDSCs in the TME are known to be the main obstacle of
   antigen presentation and CTLs function. Remarkably, a decrease in the
   percentage of M‐MDSCs (Ly6C^high Ly6G^−) was observed within the spleen
   after PDNPs treatment, indicating that PDNPs could effectively reduce
   M‐MDSCs and thus ameliorate the immunosuppressive microenvironment
   (Figure [135]5I,J, Supporting Information). Moreover, this enhanced
   immunity was further confirmed in tumors by immunofluorescence and
   immunohistochemical staining. As shown in Figure [136]5K and Figure
   [137]S45 (Supporting Information), the coexpression of CD86^+ CD11c^+,
   CD11c^+ CD103^+, and CD8^+ CD103^+ cell populations in tumor tissues
   exhibited a striking increase in the PDNPs group, indicating that PDNPs
   could effectively promote DCs maturation and T cell infiltration. The
   expression of CD8^+ PD‐1^+ T cells in tumors verified the effect of
   PDNPs on the enhancement of CTLs and the relief of CD8^+ T cells
   exhaustion. In addition, immunohistochemical staining and the
   corresponding quantitative analysis of CD86, CD8, CD103, CD11c, and
   MDSCs marker Arginase‐1 further confirmed the enhanced immunogenicity
   induced by PDNPs (Figure [138]5L; Figures [139]S46 and [140]S47,
   Supporting Information).

   Inspired by the results that PDNPs could effectively induce pyroptosis
   to boost antitumor immunity, we next explored whether the combination
   therapy of PDNPs plus the checkpoint inhibitor anti‐PD‐1 could amplify
   antitumor efficacy and extend survival through enhanced immune
   responses. As depicted in the treatment schedule, PDNPs were
   administrated preferentially to rejuvenate CD8^+ T cells, and anti‐PD‐1
   was injected intraperitoneally the next day to block PD‐1 in BALB/c
   mice bearing CT26 or 4T1 tumor xenografts (Figure [141]5M; Figure
   [142]S51A, Supporting Information). As expected, the combination of
   PDNPs and anti‐PD‐1 sharply enhanced the therapeutic efficiency
   compared with the single therapy modality (Figure [143]5N; Figures
   [144]S48, [145]S49, and S51B–D, Supporting Information), which might be
   ascribed to the immune‐potentiating effects of PDNPs that reinforce T
   cell responses. In addition, tumor growth was monitored using bright
   field and IVIS Lumina imaging, suggesting that the combination therapy
   dramatically extended survival compared to individual treatment alone
   (Figure [146]5P,Q; Figure S50, [147]S51E, Supporting Information).
   Collectively, PDNPs are capable of sensitizing tumors to
   anti‐PD‐1‐mediated immune checkpoint blockade therapy by enhancing
   antitumor immune response, thus exerting amplified therapeutic efficacy
   and extended survival.

3. Conclusions

   In summary, we have successfully fabricated a simple, tuneable, and
   safe supramolecular nanomedicine with multistage pH‐triggered size
   shrinkage and dePEGylation to combat cascaded drug delivery barriers
   and precisely delivery DOX to effectively elicit pyroptosis and augment
   antitumor immune response. The well‐tailored PDNPs exhibited high
   stability and stealth in blood circulation to avert nonspecific
   interactions with plasma proteins and clearance of the mononuclear
   phagocyte system, resulting in favorable pharmacokinetics and high
   tumor accumulation. A sharp change in particle size and detachment of
   PEG shielding in response to the acidic extracellular pH remarkably
   promoted extravasation from the tumor vasculature and infiltration into
   tumors. Subsequently, the second‐stage size shrinkage was initiated by
   lower acidic endolysosomes, and the ultrasmall cargo size ensured
   successful entry into the nucleus for the precise delivery of DOX. The
   cascaded pH activation of PDNPs effectively elicited GSDME‐mediated
   pyroptosis and potentiated immunogenicity, thus facilitating DC
   maturation and CD8^+ T cell infiltration, priming CTL proliferation,
   and boosting the antitumor immune cycle. In combination with anti‐PD‐1,
   PDNPs achieved amplified tumor suppression and significantly extended
   survival. PDNPs that elicit vigorous pyroptosis may be powerful yet
   promising immune adjuvants to augment checkpoint blockade‐based
   immunotherapy efficacy via generating a robust immune response.

Conflict of Interest

   The authors declare no conflict of interest.

Supporting information

   Supporting Information
   [148]Click here for additional data file.^ (17.9MB, pdf)

Acknowledgements