Abstract

   Small cell lung cancer (SCLC) is an extremely aggressive and highly
   malignant type of lung cancer that frequently develops resistance and
   recurrence following initial treatment. Paclitaxel (PTX) is a
   second‐line therapeutic option for SCLC patients with resistance to
   first‐line treatment. However, its clinical application is limited due
   to suboptimal efficacy and the risk of hypersensitivity reactions. To
   address these challenges, a novel therapeutic strategy employing a
   cationic liposome‐based biomimetic drug co‐delivery system,
   siPFKFB4/PRL[PTX]@RBCM‐cRGD, which simultaneously delivers paclitaxel
   and PFKFB4‐targeting small interfering RNA (siRNA) to SCLC cells and
   tissues is proposed. These findings demonstrate that this co‐delivery
   system can induce ferroptosis in SCLC cells, thereby enhancing their
   sensitivity to paclitaxel. Moreover, It promotes the infiltration of
   immune cells and the secretion of cytokines within the SCLC immune
   microenvironment, effectively activating anti‐tumor immunity. When
   combined with anti‐PD‐L1 antibodies, it further potentiates anti‐tumor
   immune responses. These results suggest that the biomimetic codelivery
   system not only induces ferroptosis to enhance paclitaxel efficacy but
   also reprograms the SCLC immune microenvironment, thereby potentiating
   the effects of anti‐PD‐L1 immunotherapy and providing a promising new
   therapeutic strategy for SCLC.

   Keywords: ferroptosis, immunotherapy, PFKFB4, Small cell lung cancer,
   tumor immune microenvironment
     __________________________________________________________________

   In this study, a targeting PFKFB4 biomimetic codelivery system is
   developed to improve paclitaxel efficacy by inducing ferroptosis in
   SCLC. This system enhances immune cell infiltration and cytokine
   secretion, activating anti‐tumor immunity. Combined with anti‐PD‐L1, it
   boosts immune responses, offering a novel therapeutic strategy for
   overcoming resistance and recurrence in small‐cell lung cancer.

   graphic file with name ADVS-12-2417374-g010.jpg

1. Introduction

   Lung cancer remains the leading cause of cancer‐related mortality
   worldwide.^[ [42]^1 ^] Small cell lung cancer (SCLC), an aggressive
   subtype of pulmonary malignancy, is characterized by its high degree of
   malignancy and a propensity for metastasis at the time of diagnosis.
   Consequently, SCLC patients face significantly lower survival rates and
   greater therapeutic challenges compared to those with non‐small cell
   lung cancer (NSCLC).^[ [43]^2 ^] Currently, the standard first‐line
   treatment for SCLC consists of radiotherapy combined with
   platinum‐based doublet chemotherapy.^[ [44]^3 ^] Despite an initial
   high sensitivity to chemotherapy, SCLC frequently develops resistance
   and recurs, with a median overall survival (OS) of only 4–5 months
   following second‐line chemotherapy after relapse.^[ [45]^4 ^]
   Paclitaxel (PTX), as one of the second‐line therapeutic agents for
   SCLC, has demonstrated antitumor activity in refractory and relapsed
   SCLC cases.^[ [46]^5 ^] However, its efficacy as a monotherapy remains
   suboptimal. Furthermore, the use of solvent‐based formulations
   increases the risk of hypersensitivity reactions, thereby limiting its
   clinical application in second‐line treatment for SCLC.^[ [47]^6 ,
   [48]^7 ^]

   In addition to chemotherapy, immune checkpoint inhibitors (ICIs) have
   been approved for the treatment of SCLC.^[ [49]^8 ^] However, only a
   limited subset of patients derives significant benefit from ICI
   therapy. The primary reason lies in the unique immune microenvironment
   of SCLC, which differs from other solid tumors and is characterized as
   a “cold” tumor immune microenvironment (TIME).^[ [50]^9 ^] Immunogenic
   cell death (ICD) is a form of cell death capable of activating adaptive
   immune responses.^[ [51]^8 , [52]^10 ^] In recent years,
   ferroptosis‐triggered immunogenic cell death has garnered significant
   attention.^[ [53]^11 , [54]^12 ^] The lipid peroxides generated during
   ferroptosis can induce the production of tumor‐associated antigens by
   tumor cells.^[ [55]^13 ^] Furthermore, ferroptosis promotes the release
   of damage‐associated molecular patterns (DAMPs), such as HMGB1 and ATP,
   through oxidative stress.^[ [56]^14 ^] These molecular signals recruit
   and activate dendritic cells (DCs), thereby initiating antigen
   presentation. Additionally, ferroptosis increases intracellular iron
   levels and reactive oxygen species (ROS) accumulation, triggering ICD
   and further modulating the tumor microenvironment.^[ [57]^15 ^]

   6‐Phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase 4 (PFKFB4), a
   bifunctional enzyme of the PFKFB family, critically regulates
   glycolytic flux and autophagy. Numerous studies have reported the
   overexpression of PFKFB4 in various human cancers. It enhances
   chemosensitivity in SCLC by suppressing autophagy, positioning it as a
   therapeutic target for drug‐resistant SCLC.^[ [58]^16 ^] Beyond tumor
   metabolism, PFKFB4 modulates immune responses through
   glycolysis‐dependent cytokine regulation.^[ [59]^17 ^] Notably, a
   co‐delivery system combining rapamycin, anti‐PFKFB4 siRNA, and AS1411
   aptamer demonstrated dual therapeutic and immunomodulatory effects,
   underscoring PFKFB4's potential in combinatorial cancer therapy.^[
   [60]^18 ^] However, the relationship between PFKFB4 and the immune
   microenvironment of SCLC requires further investigation.

   In summary, paclitaxel monotherapy demonstrates limited efficacy in the
   treatment of SCLC. Although novel paclitaxel formulations, such as
   paclitaxel liposomes, have reduced toxicity and hypersensitivity to
   some extent, their improvement in antitumor efficacy remains modest.
   PFKFB4 has shown potential as an antitumor target in enhancing
   chemosensitivity and modulating the immune microenvironment. Based on
   these findings, this study proposes a novel biomimetic drug delivery
   system designed to co‐deliver paclitaxel and PFKFB4‐targeting siRNA for
   the treatment of SCLC. Cationic liposomes were employed as
   nanocarriers, and their surface was coated with cRGD‐modified red blood
   cell membranes (RBCM) to enhance tumor‐targeting capability and achieve
   prolonged circulation in vivo. This biomimetic delivery system is
   expected to induce ferroptosis, improve SCLC sensitivity to paclitaxel,
   and activate immune responses, thereby enhancing antitumor efficacy and
   offering a new therapeutic strategy for SCLC.

2. Results

2.1. PFKFB4 is Associated with the Immune Microenvironment of SCLC and
Enhances Paclitaxel Sensitivity

   To investigate the expression of PFKFB4 in lung cancer, we analyzed
   data from the GEO and TCGA databases. As shown in Figure [61]1A,B and
   [62]S1A,B (Supporting Information), PFKFB4 expression was significantly
   higher in lung cancer tissues compared to normal tissues. Kaplan–Meier
   (KM) survival curves from the [63]GSE50081 and [64]GSE30219 datasets
   indicated that SCLC patients with high PFKFB4 expression have a poorer
   prognosis (Figure [65]1C).

Figure 1.

   Figure 1
   [66]Open in a new tab

   The Role of PFKFB4 in Small Cell Lung Cancer (SCLC). A) GEO database
   analysis of differentially expressed genes in lung cancer, with PFKFB4
   highlighted in the red box. B) GEO lung cancer dataset analysis shows
   that the expression level of PFKFB4 in lung cancer is significantly
   higher than in normal tissue. C) GEO lung cancer dataset analysis
   reveals that higher expression of PFKFB4 correlates with poorer overall
   survival (OS) in lung cancer patients. D,E) Immunohistochemical
   analysis of PFKFB4 expression levels in adjacent non‐tumor and tumor
   tissues from small cell lung cancer (SCLC) patients, along with
   quantitative analysis. F,G) Single‐cell RNA sequencing analysis of the
   immune microenvironment in SCLC, identifying various cell types. H,I)
   Single‐cell analysis of PFKFB4 expression in CD8+ T cells and other
   cell types, with PFKFB4 predominantly expressed in macrophages. J)
   Schematic diagram of the 3D structure of paclitaxel. K,L) Docking of
   PFKFB4 with paclitaxel, shows that ASN64 of PFKFB4 interacts with
   paclitaxel via multiple forces, with a binding energy of
   −7.8 kcal mol^−1. M) Comparison of the IC50 values of paclitaxel before
   and after knockdown of PFKFB4 in NCI‐H446 and DMS114 SCLC cell lines.
   ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.

   To further determine PFKFB4 expression in SCLC, we collected tumor and
   adjacent non‐tumor tissues from three SCLC patients and analyzed PFKFB4
   protein expression using IHC (Figure [67]1D). Quantitative analyses
   (Figure [68]1E) showed that PFKFB4 was highly expressed in SCLC
   tissues. Next, we annotated and clustered various cell populations,
   including T cells (Figure [69]1F,G), using the [70]GSE193960
   single‐cell SCLC dataset. We further annotated tumor cells using four
   markers closely associated with neuroendocrine tumors: ASCL1, NCAM1,
   INSM1, and GRP (Figure [71]S1H, Supporting Information). These tumor
   cells are highlighted with red circles in Figure [72]1G,H. The results
   in Figure [73]1H,I show that PFKFB4 is expressed in macrophages, tumor
   cells, and B cells, with the highest expression observed in
   macrophages. In addition, validation using TCGA datasets revealed a
   negative correlation between PFKFB4 and multiple immune cell types,
   including CD8+ T cells, macrophages, and dendritic cells (Figure
   [74]S1C—F, Supporting Information). These findings suggest that PFKFB4
   has multiple roles in small‐cell lung cancer, including involvement in
   tumorigenesis and the formation of an immunosuppressive
   microenvironment.

   Paclitaxel is one of the second‐line treatments for SCLC. Figure [75]1J
   shows the 3D structure of paclitaxel. Molecular docking analysis
   revealed multiple interactions between PFKFB4 and paclitaxel, such as a
   hydrogen bond between ASN64 of PFKFB4 and the ligand. These
   interactions resulted in a binding energy of −7.8 kcal mol^−1 for the
   protein‐ligand complex (Figure [76]1K,L).To verify whether PFKFB4
   affects paclitaxel sensitivity in SCLC, we knocked down PFKFB4 in
   NCI‐H446 and DMS114 SCLC cell lines (Figure [77]S2A—C, Supporting
   Information). Subsequent IC50 assays showed that the IC50 value of
   paclitaxel in NCI‐H446 cells decreased from 5.01 to 2.43 ng mL^−1, and
   in DMS114 cells from 78.57 to 41.91 µg mL^−1 (Figure [78]1M). These
   results indicate that knockdown of PFKFB4 significantly enhances SCLC
   sensitivity to paclitaxel (p < 0.05).

2.2. Modified Nanoparticles Stably and Efficiently Delivers siPFKFB4 while
Actively Targeting SCLC

   Cationic liposomes were synthesized using a thin‐film dispersion
   method, where DOTAP was utilized to confer a positive charge to PRL,
   enabling siRNA loading via electrostatic interactions. Red blood cell
   membranes (RBCM) were extracted using hypotonic lysis, followed by
   co‐extrusion and ultrasonic homogenization to obtain cRGD‐modified RBC
   vesicles. Finally, co‐extrusion of the liposomes and vesicles resulted
   in the siPFKFB4/PRL[PTX]@RBCM‐cRGD nanoparticles. The images, particle
   sizes, and zeta potentials of all nanoparticles used in this study are
   presented in Figure [79]S3 (Supporting Information) and Figure [80]2B.
   The siPFKFB4/PRL[PTX]@RBCM‐cRGD nanoparticles exhibited a particle size
   of ≈176.60 nm and a zeta potential of 16.6 mV (Figure [81]2A,B). The
   successful encapsulation of siRNA and RBC vesicles was confirmed by a
   significant decrease in zeta potential compared to blank nanoparticles
   (PRL) and other formulations. Figure [82]2C displays transmission
   electron microscopy (TEM) images of the blank liposomes (PRL),
   PTX‐loaded liposomes (PRL[PTX]), RBCM‐cRGD vesicles, and
   siPFKFB4/PRL[PTX]@RBCM‐cRGD nanoparticles. The pH‐responsive behavior
   of siPFKFB4/PRL[PTX]@RBCM‐cRGD was also evaluated. The drug release
   profile under simulated physiological (pH 7.4) and acidic (pH 6.5 and
   5.0) conditions at 37 °C is shown in Figure [83]2D. The results
   indicated enhanced drug release in acidic environments, particularly at
   pH 5.0 (mimicking intracellular/lysosomal conditions), where the 48 h
   release rate reached 56.15 ± 1.22%.

Figure 2.

   Figure 2
   [84]Open in a new tab

   Synthesis and Characterization of siPFKFB4/PRLPTX@RBCM‐cRGD. A)
   Particle size distribution of siPFKFB4/PRL[PTX]@RBCM‐cRGD. B) Zeta
   potential of red blood cell membrane vesicles, siRNA, and various
   nanoparticles. C) Electron microscopy images (200 nm) of empty liposome
   PRL, PTX‐loaded liposome PRLPTX, RBCM‐cRGD, and
   siPFKFB4/PRL[PTX]@RBCM‐cRGD. D) In vitro release rates of paclitaxel
   under different pH conditions (pH = 5.0, pH = 6.5, pH = 7.4). E) Gel
   retardation assay showing the binding of siRNA with cationic liposomes
   at different mass ratios. F) Agarose gel electrophoresis results
   indicate complete binding of siRNA and cationic liposomes in
   siPFKFB4/PRL[PTX]@RBCM‐cRGD. G,H) SDS‐PAGE and Western blot analysis of
   protein retention and CD47 expression in RBCM, RBCM‐cRGD, and
   siPFKFB4/PRL[PTX]@RBCM‐cRGD. I,J) Cytotoxicity of
   siPFKFB4/PRL[PTX]@RBCM‐cRGD in NCI‐H446 and DMS114 SCLC cell lines.
   K,L) Uptake of siNC/PRL and siNC/PRL@RBCM‐cRGD in NCI‐H446 and DMS114
   cells at different time points (6, 12, 24 h). Scale bars: 50 µm.

   To optimize siRNA loading onto cationic liposomes, a gel retardation
   assay was performed to determine the optimal mass ratio. As shown in
   Figure [85]2E, complete binding was achieved at a 1:10 mass ratio, and
   successful siRNA encapsulation was further confirmed in Figure [86]2F.
   SDS‐PAGE analysis demonstrated that RBCM proteins were preserved during
   nanoparticle preparation (Figure [87]2G), while Western blot analysis
   confirmed the intact retention of CD47 protein (Figure [88]2H). The
   cytotoxicity of the nanoparticles was evaluated in NCI‐H446 and DMS114
   SCLC cell lines using the CCK‐8 assay. Figure [89]2I,J show that cell
   viability remained above 85% at nanoparticle concentrations up to
   100 µg mL^−1, indicating high biocompatibility and biosafety. The
   tumor‐targeting capability of RBCM‐cRGD‐modified nanoparticles was
   assessed by comparing cellular uptake in SCLC cells. Over time, the
   fluorescence intensity of nanoparticles within cells increased
   (Figure [90]2K,L). At equivalent time points, siNC/PRL@RBCM‐cRGD
   demonstrated stronger intracellular fluorescence compared to siNC/PRL,
   confirming that RBCM‐cRGD enhanced tumor‐targeting capacity.
   Additionally, the uptake of nanoparticles by RAW264.7 macrophages was
   evaluated. Figure [91]3A shows that siNC/PRL@RBCM‐cRGD was less
   phagocytosed by macrophages than siNC/PRL, further demonstrating that
   RBCM‐cRGD reduced macrophage‐mediated clearance and enabled prolonged
   circulation in vivo. This feature ensures the nanoparticles'
   functionality within the tumor microenvironment, enhancing their
   therapeutic potential.

Figure 3.

   Figure 3
   [92]Open in a new tab

   Antitumor Activity of The Biomimetic Codelivery System In Vitro. A)
   Uptake of siNC/PRL and siNC/PRL@RBCM‐cRGD by RAW264.7 cells after 24 h.
   B) CCK‐8 assay assessing the cell viability of NCI‐H446 and DMS114 SCLC
   cells in groups G1‐G5 (G1: PRL@RBCM‐cRGD, G2: PTX, G3:
   siNC/PRL@RBCM‐cRGD, G4: siPFKFB4/PRL@RBCM‐cRGD, G5:
   siPFKFB4/PRL[PTX]@RBCM‐cRGD). C) Live/dead cell staining of NCI‐H446
   and DMS114 cells treated with G1‐G5 groups (magnification: 400×, scale
   bars: 50 µm.). D–F) Clonogenic assay, EdU incorporation assay, and PCNA
   immunofluorescence staining to evaluate the proliferative capacity of
   cells in G1‐G5 groups. G,H) Quantification of live/dead cell staining,
   clonogenic assay, EdU incorporation, and PCNA immunofluorescence (n =
   3). The concentration of PTX was 2.5 ng mL^−1 for NCI‐H446 and
   35 µg mL^−1 for DMS114. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.

   To further investigate the colloidal stability of positively charged
   nanoparticles under physiological conditions, we conducted in vitro
   stability assessments in simulated serum environments over a 7‐day
   period. Dynamic light scattering analysis revealed that the
   nanoparticles maintained their structural integrity in both
   phosphate‐buffered saline (PBS) and fetal bovine serum (FBS), with no
   statistically significant variation in hydrodynamic diameter observed
   throughout the experimental duration. As demonstrated in Figure [93]S3
   (Supporting Information), the nanoparticle size distribution remained
   stable regardless of the dispersion medium, suggesting effective
   resistance against protein adsorption and aggregation in biological
   fluids. This sustained stability profile in serum‐containing conditions
   indicates promising potential for systemic circulation applications.

2.3. The Biomimetic Codelivery System Demonstrates Potent Cytotoxic Activity
against SCLC

   To evaluate the antitumor efficacy of the biomimetic codelivery system,
   we conducted functional assays on two SCLC cell lines, NCI‐H446 and
   DMS114. The CCK‐8 assay and live‐dead staining were first used to
   assess cell viability and cytotoxicity. Results from Figure [94]3B
   revealed a significant reduction in cell viability in the
   siPFKFB4/PRL[PTX]@RBCM‐cRGD treatment group compared to other groups.
   Live‐dead staining further confirmed that siPFKFB4/PRL[PTX]@RBCM‐cRGD
   exhibited stronger cytotoxicity than PTX alone(Figure [95]3C,G). To
   investigate cell proliferation, we performed colony formation, EdU, and
   PCNA immunofluorescence assays across the G1‐G5 groups. Consistently,
   the experimental results and quantitative analyses demonstrated that
   the biomimetic codelivery system effectively suppressed SCLC cell
   proliferation (Figure [96]3D–F,H–J). These findings indicate that the
   biomimetic codelivery system exerts strong in vitro tumor‐killing
   effects.

2.4. Co‐Delivery of PTX and siPFKFB4 via Nanocarriers Amplifies Ferroptosis
in SCLC

   The superior antitumor efficacy of the co‐delivery of PTX and siPFKFB4
   compared to PTX alone prompted further mechanistic investigations.
   Transmission electron microscopy revealed mitochondrial shrinkage and
   reduced mitochondrial cristae in G5‐treated cells, hallmark features of
   ferroptosis (Figure [97]4A). Next, we assessed the expression levels of
   ferroptosis‐related proteins in the treated cell groups. Western blot
   analysis showed that siPFKFB4/PRL[PTX]@RBCM‐cRGD treatment
   significantly reduced xCT levels and upregulated ACSL4 expression in
   both cell lines (Figure [98]4B,C; Figure [99]S4A,B, Supporting
   Information). GPX4 immunofluorescence staining and quantitative
   analysis indicated that GPX4 expression was markedly elevated in the G5
   group compared to other groups (p< 0.05, Figure [100]4G,H; Figure
   [101]S5B, Supporting Information). Reactive oxygen species (ROS) levels
   were also significantly altered across treatment groups, with the
   highest ROS levels observed in cells treated with
   siPFKFB4/PRL[PTX]@RBCM‐cRGD (Figure [102]4D; Figure [103]S5A,
   Supporting Information). Additionally, malondialdehyde (MDA) and Fe^2+
   levels were markedly increased in the G5 group compared to the G2 group
   (Figure [104]4E,F). These results collectively demonstrate that the
   co‐delivery of PTX and siPFKFB4 promotes ferroptosis in SCLC cells,
   thereby enhancing their sensitivity to paclitaxel. To further clarify
   the relationship between the co‐delivery system and ferroptosis, we
   treated SCLC cells with a combination of the ferroptosis inhibitor
   Fer‐1 and the co‐delivery system, observing the changes in cell
   proliferation. The results presented in Figure [105]4I,J reveal that
   both the CCK‐8 and colony formation assays indicate that Fer‐1 alone
   does not affect the proliferation capacity of SCLC cells. However, when
   combined with the co‐delivery system (G5 group), Fer‐1 partially
   mitigates the inhibitory effect of the co‐delivery system on SCLC
   proliferation. These findings suggest that the co‐delivery system
   indeed enhances the role of ferroptosis in SCLC cells.

Figure 4.

   Figure 4
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   Synergistic Amplification of Ferroptosis by The Biomimetic Codelivery
   System to Inhibit SCLC. A) Electron microscopy images of NCI‐H446 and
   DMS114 cells treated with G1 and G5 group nanoparticles (left: 5 µm,
   right: 500 nm). B,C) Western blot analysis of xCT and ACSL4 expression
   in NCI‐H446 cells (internal reference: HSP90 and GAPDH). D)
   Intracellular ROS levels in NCI‐H446 and DMS114 cells treated with
   different groups. E,F) Intracellular levels of MDA and iron ions in
   NCI‐H446 and DMS114 cells under different treatments (n = 3). ^* p
   < 0.05, ^** p < 0.01, ^*** p < 0.001. G,H) Immunofluorescence detection
   of GPX4 expression in NCI‐H446 and DMS114 cells in G1‐G5 groups. I) The
   CCK‐8 assay was employed to assess the impact of the combined treatment
   with Fer‐1 and siPFKFB4/PRL[PTX]@RBCM‐cRGD on the proliferative
   capacity of SCLC cells. J) The colony formation assay was performed to
   evaluate the anti‐proliferative effects of the combined treatment with
   Fer‐1 and the co‐delivery system in SCLC cells.

2.5. Nanoparticle Targeting of PFKFB4 Promotes M1 Polarization and Activates
DC‐Induced Immune Responses In Vitro

   Previous results indicated that PFKFB4 expression is negatively
   correlated with the infiltration of most immune cells in SCLC. To
   validate this, we utilized immortalized murine macrophages (RAW264.7)
   and induced bone marrow‐derived macrophages (BMDM) and dendritic cells
   (BMDC). These cells were co‐cultured with nanoparticles and murine SCLC
   cells to simulate the tumor microenvironment and explore the role of
   the biomimetic codelivery system in immune modulation (Figure [107]S6,
   Supporting Information). We first assessed markers of M1 and M2
   macrophage polarization. Flow cytometry revealed that MHC‐II expression
   in BMDM and RAW264.7 cells increased progressively from G1 to G5
   groups, while CD206 expression decreased, suggesting a trend toward M1
   polarization (Figure [108]5A–D; Figure [109]S7, Supporting
   Information). RT‐qPCR results further confirmed this shift, as
   M1‐associated chemokine IL1β, CXCL10 and TNF‐α levels were
   significantly elevated in the siPFKFB4/PRL[PTX]@RBCM‐cRGD group, while
   M2‐associated markers MGL2, and MRC1 were significantly downregulated
   (p < 0.05, Figure [110]5E). Immunofluorescence analysis showed
   increased expression of CD86 and reduced ARG1 levels in the G5 group
   compared to other groups, further supporting M1 polarization
   (Figure [111]5G,H). Next, we evaluated the maturation of BMDCs after
   co‐culture via flow cytometry. The proportion of CD80 and CD86
   double‐positive DCs increased significantly across G1‐G5 groups, with
   the highest levels observed in the G5 group (Figure [112]5I). These
   findings demonstrate that the biomimetic codelivery system strongly
   promotes DC maturation. Collectively, the results indicate that the
   nanoparticle‐mediated targeting of PFKFB4 is closely associated with
   the SCLC immune microenvironment and exhibits robust immune activation
   capabilities in vitro.

Figure 5.

   Figure 5
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   Status of Different Immune Cells after Co‐culture with
   siPFKFB4/PRL[PTX]@RBCM‐cRGD and RPM Cells. A,B) Flow cytometry analysis
   of MHC‐II expression, a marker for M1 polarization, in bone
   marrow‐derived macrophages (BMDM) and RAW264.7 cells after co‐culture.
   C,D) Flow cytometry analysis of CD206 expression, a marker for M2
   polarization, in BMDM and RAW264.7 cells following co‐culture. E)
   RT‐qPCR analysis of M1 polarization markers CXCL10, TNF‐α, IL‐1β, and
   M2 polarization markers MGL‐2 and MRC1 mRNA expression levels in BMDM
   after co‐culture (n = 3). ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.
   F–H) Immunofluorescence analysis of Arg1 and CD86 expression in BMDM
   cells co‐cultured with different nanoparticles and RPM cells, with
   quantitative analysis (n = 3). I,J) Percentage and quantitative
   analysis of CD80 and CD86 double‐positive dendritic cells (DCs) in
   G1‐G5 co‐culture groups.

2.6. Nanoparticle Co‐Delivery System Activates the JAK‐STAT Pathway to
Modulate the Immune Microenvironment in SCLC

   To elucidate the mechanisms underlying the antitumor effects of the
   nanoparticle co‐delivery system and its regulation of the SCLC immune
   microenvironment, we treated NCI‐H446 and DMS114 cells with
   PRL@RBCM‐cRGD and siPFKFB4/PRL[PTX]@RBCM‐cRGD, followed by
   transcriptomic analysis. Volcano plots highlighted differentially
   expressed genes (DEGs) after nanoparticle treatment (Figure [114]6A;
   Figure [115]S8A, Supporting Information). Gene Set Enrichment Analysis
   (GSEA) revealed enrichment of pathways associated with
   cytokine‐receptor interactions (Figure [116]6B; Figure [117]S8B,
   Supporting Information). By intersecting DEGs with ferroptosis‐related
   genes, we identified nine genes potentially critical for ferroptosis
   induction by the system: ACSL3, AKT1S1, ATM, CD44, CHAC1, CREB5, HMOX1,
   NEDD4L, and NR4A1 (Figure [118]6C,D). Expression changes of these genes
   in the G1 and G5 groups were consistent with patterns observed during
   ferroptosis, further corroborating that the nanoparticle co‐delivery
   system enhances PTX sensitivity and antitumor efficacy by promoting
   ferroptosis. Functional enrichment analysis (Figure [119]6E–H) linked
   nanoparticle activity to cytokine‐receptor interactions and DNA‐binding
   pathways. Given the established role of the JAK‐STAT pathway in
   mediating cytokine effects,^[ [120]^19 , [121]^20 ^] we assessed the
   expression of key JAK‐STAT proteins in BMDCs and BMDMs co‐cultured with
   nanoparticles and SCLC cells. As shown in Figure [122]6I–L, expression
   of phosphorylated JAK1 (p‐JAK1) and phosphorylated STAT1 (p‐STAT1) was
   significantly elevated in immune cells after co‐culture, particularly
   in the siPFKFB4/PRL[PTX]@RBCM‐cRGD group. These findings suggest that
   JAK‐STAT pathway activation is a critical mechanism by which the
   biomimetic codelivery System modulates the SCLC immune
   microenvironment.

Figure 6.

   Figure 6
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   Mechanistic Exploration of The Biomimetic Codelivery System in
   Inhibiting SCLC. A) Volcano plot of differentially expressed genes
   after transcriptome sequencing of NCI‐H446 cells treated with RBCM‐cRGD
   and siPFKFB4/PRL[PTX]@RBCM‐cRGD. B) Gene Set Enrichment Analysis
   (GSEA). C) Venn diagram showing the intersection of differentially
   expressed genes in NCI‐H446 and DMS114 cells treated with
   siPFKFB4/PRL[PTX]@RBCM‐cRGD and ferroptosis‐related genes. D) Heatmap
   of the expression of 9 selected ferroptosis‐related genes in NCI‐H446
   cells. E–H) KEGG and GO pathway enrichment analysis of the common
   differentially expressed genes between NCI‐H446 and DMS114 cells. I,J)
   Western blot analysis and quantitative analysis of JAK‐STAT pathway
   proteins in BMDC cells treated with RBCM‐cRGD and
   siPFKFB4/PRL[PTX]@RBCM‐cRGD (n = 3). K,L) Western blot analysis and
   quantitative analysis of JAK‐STAT pathway proteins in BMDM cells
   treated with RBCM‐cRGD and siPFKFB4/PRL[PTX]@RBCM‐cRGD (n = 3).

2.7. The Biomimetic Codelivery System Shows Excellent Targeting of SCLC
Tumors with Minimal Toxicity

   Given the promising antitumor efficacy and biosafety demonstrated by
   the biomimetic codelivery System in vitro, we proceeded to evaluate its
   therapeutic potential in vivo. A murine subcutaneous SCLC model was
   established using RPM cells. Mice were intravenously injected with
   Cy5.5‐labeled PRL or PRL@RBCM‐cRGD nanoparticles, and in vivo,
   fluorescence imaging was conducted at 2, 12, and 24 h to monitor
   nanoparticle distribution. As shown in Figure [124]7A, both PRL and
   PRL@RBCM‐cRGD initially accumulated in the heart and liver but
   gradually localized to the tumor region over time. By 24 h,
   fluorescence signals were primarily concentrated in the liver, kidneys,
   and tumor sites, with PRL@RBCM‐cRGD showing significantly enhanced
   tumor accumulation compared to PRL (Figure [125]7B,C). These findings
   suggest that RBCM‐cRGD imparts enhanced tumor‐targeting capability to
   nanoparticles. To assess the biocompatibility of
   siPFKFB4/PRL[PTX]@RBCM‐cRGD, tumor‐bearing mice were injected with
   formulations identical to those used in vitro studies, and potential
   organ damage and hematological changes were evaluated. As shown in
   Figure [126]7D, neither PTX nor siPFKFB4/PRL[PTX]@RBCM‐cRGD caused
   significant organ damage. Liver, kidney, and cardiac functions, along
   with hematological indices, showed no notable differences among the
   five groups (Figure [127]7G–J; Figure [128]S9B—F, Supporting
   Information), demonstrating the favorable biocompatibility of the
   biomimetic codelivery System.

Figure 7.

   Figure 7
   [129]Open in a new tab

   Biocompatibility and Tumor Targeting Ability of The Biomimetic
   Codelivery System in Vivo. A) In vivo imaging of C57 mice after
   subcutaneous tumor formation and tail vein injection of PRL and
   PRL@RBCM‐cRGD. Nanoparticle uptake was observed at 2, 12, and 24 h (n =
   3). B,C) After 24 h of tail vein injection, mice were humanely
   euthanized, and imaging was performed on the heart, liver, spleen,
   lungs, kidneys, and tumor tissues to observe nanoparticle uptake,
   followed by quantitative analysis. D,E) Hematoxylin and eosin (H&E)
   staining of heart, liver, spleen, lungs, kidneys, and tumor tissues
   after tail vein injection of G1‐G5 nanoparticles or drugs (G1:
   PRL@RBCM‐cRGD, G2: PTX, G3: siNC/PRL@RBCM‐cRGD, G4:
   siPFKFB4/PRL@RBCM‐cRGD, G5: siPFKFB4/PRL[PTX]@RBCM‐cRGD). Scale bars:
   100 µm. F) Immunohistochemical analysis of Ki67 expression in tumor
   tissues from G1‐G5 groups. G–J) Blood samples from G1‐G5 groups were
   collected to assess liver function (AST), kidney function (BUN), heart
   function (CK‐MB), and blood routine (HGB) (n = 3). ^* p < 0.05, ^** p
   < 0.01, ^*** p < 0.001.

2.8. Potent In Vivo Antitumor Efficacy of the Nanoparticle Co‐Delivery System
Against SCLC

   The in vivo antitumor efficacy of the nanoparticle co‐delivery system
   was subsequently evaluated. Tumor‐bearing mice were divided into five
   groups for further experiments, as outlined in Figure [130]8A. PTX was
   administered at 10 mg kg^−1 weekly for a total of four doses. Tumor
   volume was significantly larger in Group G1 compared to Group G5, where
   tumor volume was smallest (Figure [131]8B). Although PTX monotherapy
   reduced tumor size, the nanoparticle co‐delivery system exhibited
   superior antitumor efficacy (Figure [132]8C,E). Mouse body weight
   remained stable across all groups during treatment (Figure [133]8D),
   indicating the therapeutic regimen's safety. These results highlight
   the enhanced efficacy of combining siPFKFB4 with PTX in SCLC treatment.
   Post‐treatment tumor tissues were subjected to histological examination
   via hematoxylin‐eosin (HE) staining and Ki67 immunohistochemistry. HE
   staining revealed progressive increases in tumor necrosis and
   intercellular gaps from G1 to G5 (Figure [134]7E). Ki67 expression, an
   indicator of proliferation, declined progressively, with the lowest
   levels observed in G5 (Figure [135]7F; Figure [136]S9A, Supporting
   Information). Immunofluorescence analysis further confirmed these
   findings; siPFKFB4/PRL[PTX]@RBCM‐cRGD‐treated tumors exhibited
   significant reductions in GPX4 expression and elevated reactive oxygen
   species (ROS) levels (Figure [137]8F,H), consistent with in vitro
   observations. These results suggest that the biomimetic codelivery
   system induces ferroptosis in SCLC cells.

Figure 8.

   Figure 8
   [138]Open in a new tab

   In Vivo Inhibition of SCLC by The Biomimetic Codelivery System. A)
   Treatment strategy for RPM tumor‐bearing mice. B) Tumor growth
   differences in SCLC after treatment with G1‐G5 nanoparticles (n = 5 per
   group). C) Tumor volume differences in mice across different treatment
   groups. D) Body weight changes in mice during the treatment period. E)
   Tumor weight at the end of treatment in different groups. F–I)
   Immunofluorescence analysis of GPX4, HMGB1 expression, ROS levels, and
   quantitative analysis in different treatment groups. ^* p < 0.05, ^** p
   < 0.01, ^*** p < 0.001.

2.9. PFKFB4‐Targeted Nanoparticle Co‐Delivery System: Immune Activation and
Synergy with PD‐L1 Antibodies In Vivo

   Previous in vitro studies demonstrated the role of the PFKFB4‐targeted
   nanoparticle co‐delivery system in modulating the SCLC immune
   microenvironment. In vivo experiments further validated this
   phenomenon. As shown in Figure [139]8G, HMGB1 expression in the G5
   group was 6.9 fold higher than in the G1 group, indicating significant
   immunogenic cell death (ICD) induction. The PFKFB4‐targeted
   nanoparticle co‐delivery system increased the proportion of M1
   macrophages (up to 56.7%) while decreasing M2 macrophages (from 50.6%
   to 27.0%), resulting in an M1/M2 ratio of 1.92 (Figure [140]9A; Figure
   [141]S11, Supporting Information). The maturation of dendritic cells
   (DCs), a critical component of antigen presentation, also improved,
   with mature DCs accounting for 40.5% of total DCs (Figure [142]9B).
   CD8+ T cells, essential for antitumor immunity, increased to 32.0%,
   with a significant rise in GZMA+ CD8+ T cells (Figure [143]9C,D).
   Immunofluorescence further confirmed enhanced CD8α and CD86 expression
   in the G5 group compared to G1 (Figure [144]9E,F). Cytokine secretion,
   including IFN‐γ, IL‐2, IL‐12, and TNF‐α, was also markedly elevated in
   the G5 group (Figure [145]9G–J), collectively demonstrating robust
   immune activation.

Figure 9.

   Figure 9
   [146]Open in a new tab

   Tumor Immune Response Induced by The Biomimetic Codelivery System. A)
   Flow cytometry analysis of the proportion of M2 macrophages in tumor
   tissues from G1‐G5 groups and quantitative analysis. B) Differences in
   the maturation of dendritic cells (DCs) in tumor tissues from G1‐G5
   groups and quantitative analysis. C) Proportion of CD8+ T cells in
   tumor tissues from G1‐G5 groups and quantitative analysis. D)
   Proportion of cytotoxic T cells in tumor tissues from G1‐G5 groups and
   quantitative analysis. E,F) Double immunofluorescence staining of CD8a
   and CD86 expression in tumor tissues from G1 (PRL@RBCM‐cRGD) and G5
   (siPFKFB4/PRL[PTX]@RBCM‐cRGD) groups, along with quantitative analysis
   (n = 3). G–J) Serum ELISA analysis of different cytokine levels in
   G1‐G5 groups. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.

   Interestingly, PD‐L1 expression in G5 group tumor tissues was higher
   than in G1, prompting further exploration of combined therapy with
   PD‐L1 antibodies (Figure [147]10A; Figure [148]S12D, Supporting
   Information). As shown in Figure [149]10C–G,
   siPFKFB4/PRL[PTX]@RBCM‐cRGD, and PD‐L1 antibodies each achieved certain
   antitumor effects as monotherapies; however, their combination
   significantly enhanced tumor suppression. Notably, no significant
   changes in mouse body weight (Figure [150]10H) or liver and kidney
   function (Figure [151]10I,J; Figure [152]S12A—C, Supporting
   Information) were observed, underscoring the safety of this combined
   approach. Together, these results highlight the synergistic potential
   of the biomimetic codelivery system with PD‐L1 antibodies in enhancing
   antitumor efficacy.

Figure 10.

   Figure 10
   [153]Open in a new tab

   Combination Therapy of siPFKFB4/PRL[PTX]@RBCM‐cRGD and Anti‐PD‐L1
   Antibody Inhibits SCLC. A) PD‐L1 expression levels in tumor tissues
   from tumor‐bearing mice treated with PRL@RBCM‐cRGD and
   siPFKFB4/PRL[PTX]@RBCM‐cRGD. B) Combination treatment with
   siPFKFB4/PRL[PTX]@RBCM‐cRGD and anti‐PD‐L1 antibody. C–G) Tumor volume
   and tumor weight differences in mice from different treatment groups
   (PRL@RBCM‐cRGD+vector, PRL@RBCM‐cRGD+anti‐PD‐L1,
   siPFKFB4/PRL[PTX]@RBCM‐cRGD+vector,
   siPFKFB4/PRL[PTX]@RBCM‐cRGD+anti‐PD‐L1, n = 5 per group). H) Body
   weight changes in mice during the combination therapy process (n = 5
   per group). I,J) Liver function indicators in mice from different
   groups (n = 3).

3. Discussion

   As one of the drug options following first‐line resistance in SCLC
   patients, paclitaxel has not become the preferred choice for
   second‐line treatment. The main reasons are its suboptimal efficacy in
   SCLC treatment and the hypersensitivity and toxicity associated with
   conventional paclitaxel due to the use of solvents. To enhance the
   efficacy of paclitaxel and reduce solvent‐induced hypersensitivity
   reactions, novel drug delivery technologies such as liposomes,
   microspheres, and nanoparticles have been proposed for their
   application in SCLC treatment.^[ [154]^7 ^] Additionally, exploring new
   targets to increase paclitaxel sensitivity in SCLC is another approach
   to enhancing its therapeutic efficacy. As shown in the results in
   Figure [155]1, SCLC patients with high PFKFB4 expression have a poorer
   prognosis, suggesting that PFKFB4 plays a critical role in the
   development and progression of SCLC. Single‐cell data analysis and
   immune cell infiltration analysis indicate that PFKFB4 is predominantly
   expressed in macrophages within the immune microenvironment of SCLC.
   Furthermore, the expression level of PFKFB4 is negatively correlated
   with CD8+ T cells, macrophages, dendritic cells, and other cell types
   integral to antitumor immunity. This suggests that PFKFB4 may regulate
   the immune microenvironment of SCLC and primarily contribute to the
   formation of an immunosuppressive microenvironment. These findings
   indicate that PFKFB4 has potential as a novel therapeutic target for
   SCLC. Moreover, the knockdown of PFKFB4 in two SCLC cell lines
   significantly reduced the IC50 of paclitaxel, demonstrating that PFKFB4
   knockdown markedly enhances the sensitivity of SCLC to paclitaxel.
   Targeting or inhibiting PFKFB4 in combination with paclitaxel treatment
   may yield improved antitumor effects in SCLC.

   In this study, a pH‐sensitive cationic liposome was employed as a
   nanocarrier to co‐deliver PTX and siPFKFB4, with the outermost layer
   coated with red blood cell membrane vesicles conjugated with cRGD. The
   nanoparticles had an average diameter of 176.60 nm, and their
   successful encapsulation was confirmed by zeta potential measurements
   and electron microscopy images (Figure [156]2B,C). Red blood cell
   membrane vesicles, serving as a natural coating, are widely used in
   drug delivery studies due to their inherent biocompatibility, enhanced
   material stability, and ability to evade immune clearance.^[ [157]^21 ,
   [158]^22 , [159]^23 ^] Consistent with previous studies, the retention
   of proteins on the red blood cell membranes, the expression of CD47,
   and uptake experiments using RAW264.7 cells suggest that the
   encapsulation of the codelivery system by red blood cell membranes
   enables the nanoparticles to evade macrophage phagocytosis and achieve
   prolonged circulation in vivo. Cytotoxicity assays, hematoxylin and
   eosin (H&E) staining of various organs, and blood biochemical analysis
   demonstrate that the nanoparticle co‐delivery system exhibits high
   biosafety both in vitro and in vivo. Conventional nanodrugs primarily
   rely on the enhanced permeability and retention (EPR) effect for
   passive tumor accumulation.^[ [160]^24 , [161]^25 ^] To enable active
   tumor targeting, cRGD, a commonly used targeting peptide for
   nanomaterial modification,^[ [162]^26 , [163]^27 ^] was conjugated to
   the red blood cell membrane vesicles. Results from Figure [164]2K,L,
   and [165]7A,B show that RBCM‐cRGD improves tumor accumulation of the
   nanoparticles and enhances drug uptake by tumor cells. Collectively,
   these findings indicate that the biomimetic codelivery system exhibits
   excellent safety and high tumor‐targeting capability, showing promise
   for future clinical applications in SCLC treatment.

   Notably, nanoparticle‐mediated targeting of PFKFB4 demonstrated potent
   antitumor efficacy in both in vitro and in vivo experiments. Based on
   the aforementioned results, the enhanced tumor‐killing effect of the
   system compared to PTX or PFKFB4 inhibition alone can be attributed to
   the increased sensitivity of SCLC to PTX upon PFKFB4 downregulation.
   Ferroptosis, a non‐apoptotic form of cell death driven by
   iron‐dependent lipid peroxidation, is often accompanied by aberrant
   expression of iron metabolism‐related molecules such as GPX4^[ [166]^28
   , [167]^29 ^] and xCT.^[ [168]^30 ^] Chemoresistant tumor cells
   frequently exhibit high sensitivity to ferroptosis, suggesting its
   potential to enhance chemotherapy sensitivity and reverse drug
   resistance.^[ [169]^31 ^] Inducing ferroptosis has been reported to
   improve PTX sensitivity in several cancer types, including
   triple‐negative breast cancer,^[ [170]^32 , [171]^33 ^] esophageal
   cancer,^[ [172]^34 ^] and hepatocellular carcinoma.^[ [173]^35 ^]
   Similarly, our electron microscopy findings revealed significant
   ferroptotic features in SCLC cells treated with the nanoparticle
   co‐delivery system. Ferroptosis‐related proteins such as GPX4 and xCT
   were downregulated, whereas ACSL4 expression was markedly upregulated.
   Treatment also resulted in increased intracellular levels of MDA and
   ROS, along with iron ion accumulation (Figure [174]4E,F). Elevated ROS
   levels and decreased GPX4 expression were further validated in tumor
   tissues from treated mice (Figure [175]8F,G). Furthermore, in both the
   CCK‐8 and colony formation assays, we observed that the ferroptosis
   inhibitor Fer‐1 partially mitigates the inhibitory effect of the
   co‐delivery system on the proliferation of SCLC cells
   (Figure [176]4I,J). These results collectively indicate that the
   targeting PFKFB4 nanoparticle co‐delivery system induces ferroptosis in
   SCLC cells by suppressing PFKFB4, thereby enhancing the antitumor
   efficacy of PTX.

   It is well established that the limited clinical benefit of ICIs in
   SCLC patients is largely attributable to the unique immunosuppressive
   tumor microenvironment characteristic of this malignancy. The immune
   microenvironment of SCLC tumors is generally acidic, enriched with
   glutathione and hydrogen peroxide,^[ [177]^36 ^] in addition to sharing
   key pathophysiological features common to solid tumors such as
   hypoxia.^[ [178]^37 ^] Moreover, SCLC exhibits low levels of
   tumor‐infiltrating lymphocytes,^[ [179]^38 ^] significantly reduced
   expression of programmed PD‐L1,^[ [180]^39 , [181]^40 ^] deficient
   expression of major histocompatibility complex class I (MHC‐I)
   molecules,^[ [182]^41 ^] and absent expression of MHC class II
   proteins,^[ [183]^42 ^] all of which contribute to its classification
   as a cold TIME. These factors pose substantial challenges to the
   efficacy of ICIs in SCLC treatment. Consequently, there is a pressing
   need to identify novel therapeutic targets or develop advanced drug
   delivery systems to remodel the SCLC immune microenvironment and enable
   more patients to benefit from immunotherapy.

   Recent evidence suggests that inducing ferroptosis not only inhibits
   tumor growth but also activates the immune system and enhances
   immunotherapy responses.^[ [184]^43 , [185]^44 ^] During ferroptosis,
   the tumor immune microenvironment may shift, suppressing tumor
   progression and metastasis.^[ [186]^45 ^] Furthermore,
   ferroptosis‐induced ICD exhibits potent immunostimulatory effects
   through the release of DAMPs, such as HMGB1, which activate dendritic
   cell maturation and enhance cytotoxic T lymphocyte infiltration,
   thereby bridging oxidative lipid peroxidation with antitumor
   immunity.^[ [187]^46 ^] Our results demonstrate that the elevated HMGB1
   expression observed in mouse tumor tissues following treatment with
   engineered lipid nanoparticles indicates that immunogenic cell death
   occurred in SCLC cells. Furthermore, in vitro co‐culture experiments
   simulating the SCLC immune microenvironment, combined with in vivo
   studies, revealed significant immune modulation. Focused analysis of
   tumor‐associated macrophage (TAM) polarization, dendritic cell (DC)
   maturation, and cytotoxic T‐cell infiltration showed that the
   biomimetic codelivery system shifted TAMs toward an M1 phenotype, as
   evidenced by increased CD86 and MHC‐II expression and decreased CD206
   and Arg1 expression. Additionally, cytokines such as TNF‐α were
   significantly elevated, indicating a pro‐inflammatory state. These
   findings were corroborated in vivo, with CD80/CD86 double‐positive DCs
   accounting for 30–40% of the population. Furthermore, cytotoxic T‐cell
   infiltration was significantly increased in tumor tissues from treated
   mice, indicating an activated immune microenvironment. These results
   suggest that the co‐delivery system amplifies ferroptosis‐induced ICD,
   thereby activating the microenvironment of SCLC.

   Transcriptomic sequencing and enrichment analyses revealed the
   activation of cytokine‐related pathways, particularly the JAK‐STAT
   pathway, which is known to regulate immune responses in the tumor
   microenvironment.^[ [188]^47 , [189]^48 ^] Western blot analysis
   confirmed JAK‐STAT pathway activation in DCs and macrophages,
   underscoring its critical role in reversing the immunosuppressive
   microenvironment of SCLC. Notably, PD‐L1 expression was upregulated in
   tumor tissues following treatment with PFKFB4‐targeted nanoparticles.
   Previous studies have shown that increased PD‐L1 expression enhances
   the efficacy of anti‐PD‐L1 antibodies.^[ [190]^49 , [191]^50 ^]
   Combining siPFKFB4/PRL[PTX]@RBCM‐cRGD with anti‐PD‐L1 antibodies
   resulted in synergistic therapeutic effects, significantly suppressing
   tumor growth, thereby providing a strong theoretical basis for
   combining chemotherapy with immunotherapy in clinical applications.

   While the biomimetic codelivery system demonstrated significant
   antitumor efficacy and remarkable modulation of the tumor immune
   microenvironment in SCLC, this study has certain limitations.
   Variability in product batches and the complexity of the tumor immune
   microenvironment pose challenges for its clinical translation. The
   immunomodulatory effects and potential biological interactions of
   positively charged nanoparticles in vivo warrant comprehensive
   investigation. Additionally, the co‐delivery system was not evaluated
   in multiple SCLC subtypes or patient‐derived xenograft (PDX) models.
   Further research is needed to elucidate precise regulatory mechanisms
   and address these challenges to facilitate clinical applications.

4. Conclusion

   This study proposes a biomimetic red blood cell membrane‐based
   co‐delivery system for PTX and siPFKFB4. This system demonstrates
   excellent biocompatibility and prolonged circulation in vivo, with the
   capability of actively targeting tumors and responding to the acidic
   tumor microenvironment. It enhances therapeutic sensitivity by
   promoting ferroptosis in SCLC cells. Furthermore, the PFKFB4‐targeted
   nanoparticle co‐delivery system induces immunogenic cell death in tumor
   cells, leading to the secretion of relevant cytokines and activation of
   the JAK‐STAT signaling pathway in DCs and tumor‐associated macrophages
   (TAMs). This results in the maturation of DCs, polarization of TAMs
   toward the M1 phenotype, and activation of effector T cells, thereby
   enhancing antitumor immunity. The combination of the biomimetic
   codelivery system with anti‐PD‐L1 antibodies synergistically improves
   the efficacy of antitumor immunotherapy.

5. Experimental Section

Cell Culture

   Human SCLC cell lines NCI‐H446 and DMS114, along with RAW264.7 murine
   macrophages, were purchased from Procell (Wuhan, China). The murine
   SCLC cell line RPM was derived from primary orthotopic lung tumors of
   Rb1^flox/flox, Trp53^flox/flox, and Myc^LSL/LSL (RPM) mice, obtained
   from Jackson Laboratory (#02 9971). NCI‐H446 and RPM cells were
   cultured in 1640 medium supplemented with 10% (v/v) fetal bovine serum
   (FBS) under a 5% CO₂ atmosphere at 37 °C. DMS114 and RAW264.7 cells
   were cultured in DMEM medium supplemented with 10% FBS under the same
   conditions.

Reagents and Materials

   Reagents included fetal bovine serum (Procell, Wuhan, China), DMEM and
   1640 media (Nanjing KeyGen Biotech Inc., China), ROS probes (MCE, USA),
   MDA assay kits (Solarbio, Beijing, China), paclitaxel (Rhawn,
   Guangzhou, China), Cell Counting Kit‐8 (GLPBIO, USA), and Cell Ferrous
   Iron Colorimetric Assay Kit (Elabscience, Wuhan, China). Edu assay kits
   and Live/Dead cell double‐staining kits were purchased from Abbkine
   Scientific Co., Ltd (Wuhan, China). Lipids used in nanoparticle
   preparation included 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphorylcholine
   (DPPC), cholesterol, 1,2‐dioleoyl‐3‐trimethylammonium propane (DOTAP),
   DSPE‐PEG2K‐cRGD, and
   1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐poly(2‐ethyl‐2‐oxazolin
   e) (DSPE‐PEOz).

Synthesis of siPFKFB4/PRL[PTX]@RBCM‐cRGD

   Cationic liposomes were synthesized using a thin‐film dispersion
   method. Appropriate amounts of DPPC, cholesterol, DOTAP, DSPE‐PEOz,
   Cy5.5, and PTX were dissolved in a chloroform‐methanol mixture. The
   solution was ultrasonicated for uniform mixing and transferred to a
   rotary evaporator, where it was rotated at 40 °C for 15 min to form a
   lipid film. The film was hydrated with DEPC‐treated water,
   ultrasonicated, and extruded repeatedly through 200 nm polycarbonate
   membranes using a liposome extruder (Avanti, USA) to obtain PRL[PTX].
   PRL[PTX] and siPFKFB4 were mixed in an enzyme‐free EP tube at a mass
   ratio determined by gel retardation assay, ultrasonicated, and
   incubated at room temperature for 15–20 min to allow binding, yielding
   siPFKFB4/PRL[PTX], which was stored at 4 °C.

   Mouse blood was obtained via cardiac puncture and centrifuged at
   3000 rpm at 4 °C for 20 min to remove serum. The red blood cells (RBCs)
   were washed with PBS, centrifuged, and resuspended in 0.25 × PBS at a
   1:40 volume ratio. The mixture was incubated at 4 °C for 2 h to lyse
   the RBCs, followed by high‐speed centrifugation at 12000 rpm at 4 °C
   for 20 min. The lysed RBCs were washed with 0.25 × PBS hypotonic
   solution and centrifuged to obtain pale red or white RBC membranes. The
   membranes were extruded sequentially through 800, 400, and 200 nm
   polycarbonate membranes to produce red blood cell membrane vesicles
   (RBCM). RBCM was mixed with PBS‐dissolved cRGD and ultrasonicated,
   followed by incubation at 37 °C for 20 min to obtain RBCM‐cRGD.
   Finally, siPFKFB4/PRL[PTX] and RBCM‐cRGD were mixed in an appropriate
   ratio and ultrasonicated at 100 W and 59 Hz for 5 min. The mixture was
   extruded through a liposome extruder, sequentially passing through 800,
   400, and 200 nm polycarbonate membranes to obtain
   siPFKFB4/PRL[PTX]@RBCM‐cRGD with uniform particle size.

Gel Retardation Assay

   A 2% agarose gel was prepared by dissolving 0.4 g of agarose in 20 mL
   of 0.5×TBE buffer with gentle shaking. The mixture was microwaved until
   the agarose fully dissolved. After cooling to ≈60 °C, 2 µL of Gel Red
   (Biosharp, China) was added and gently mixed before pouring into a gel
   mold. The gel was left to solidify. Samples from different groups were
   loaded into separate wells of the gel. Electrophoresis was performed at
   100 V for 20 min. The gel was imaged to assess sample migration.

SDS‐PAGE Electrophoresis and Western Blot

   For protein extraction, samples were lysed with RIPA buffer containing
   1% protease and phosphatase inhibitors. After 30 min on ice, the
   lysates were centrifuged, and the supernatant containing extracted
   proteins was collected. Protein concentrations were quantified using
   the BSA method. Equal amounts of protein were loaded onto pre‐prepared
   10% SDS‐PAGE gels for electrophoresis. SDS‐PAGE Electrophoresis: The
   gels were stained with Coomassie Brilliant Blue for 30 min and
   destained until clear protein bands were visible against a transparent
   background. Bands were visualized using a gel imaging system. Western
   Blot: Proteins were transferred to a polyvinylidene fluoride (PVDF)
   membrane, which was then blocked with skim milk for 120 min. After
   washing, the membrane was incubated with primary antibodies at 4 °C for
   16–18 h. Secondary antibodies were incubated at room temperature for
   ≈120 min. Enhanced chemiluminescence (ECL) reagents were used to detect
   protein bands, which were visualized using a chemiluminescence imaging
   system. Details of antibodies used are provided in the [192]Supporting
   Information.

Cell Viability Assay

   Cells were seeded at a density of 6 × 10^3 per well in 96‐well plates.
   After adhesion, cells were treated with PTX or nanoparticles at
   different concentrations and incubated for 24 h. A complete medium
   containing 10% CCK‐8 solution was added to each well and incubated for
   an additional 2 h. Absorbance was measured using a microplate reader.
   Each experiment was independently repeated three times.

Cellular Uptake Assay

   Cells were seeded onto confocal dishes. After adhesion, 500 µL of
   complete medium containing siNC/PRL or siNC/PRL@RBCM‐cRGD was added.
   After incubation in the dark for 6, 12, and 24 h, the dishes were
   retrieved, and cells were fixed with 4% paraformaldehyde for 20 min.
   Nuclei were stained with DAPI, and coverslips were mounted. Cellular
   uptake was visualized using a confocal microscope.

Colony Formation Assay

   Cells were seeded into six‐well plates and allowed to adhere. The cells
   were then treated with media containing nanoparticles or drugs and
   incubated for 24 h. Fresh medium was replaced every 2–3 days. Once
   visible colonies formed, cells were fixed with paraformaldehyde for 20
   min, washed with PBS, and stained with crystal violet. The stained
   plates were washed with PBS, air‐dried, and photographed.

Live/Dead Staining Assay

   Cells were seeded in confocal dishes and incubated in a CO₂ incubator
   at 37 °C for 24 h. Subsequently, 500 µL of medium containing
   nanoparticles or drugs were added, and cells were incubated for another
   24 h. After two PBS washes, 0.5 mL of staining solution was added, and
   the cells were incubated in the dark at 37 °C for 30 min. After another
   PBS wash, coverslips were mounted, and fluorescence was immediately
   observed under a fluorescence microscope.

EdU Assay

   Reagents from the EdU assay kit were prepared in advance. Cells were
   seeded into confocal dishes and treated with nanoparticles or drugs for
   24 h. A 2× EdU solution, prewarmed to 37 °C, was diluted to 1× final
   concentration in the medium and added to the cells. After 2 h of
   incubation, cells were fixed, washed, and permeabilized. A 100 µL
   Click‐iT reaction mixture was added to each sample, and cells were
   incubated at room temperature in the dark for 30 min. After washing
   with 1× BSA wash solution for 5 min, nuclei were stained, coverslips
   were mounted, and fluorescence was observed under a fluorescence
   microscope.

ROS Detection

   The ROS detection probe H[2]DCFDA was dissolved in DMSO to prepare a
   10 mM stock solution, which was further diluted before use. Cells were
   incubated with PBS containing 5 µM H[2]DCFDA at 37 °C in the dark for
   30 min. After washing with PBS, fluorescence was observed using a
   confocal microscope.

MDA Detection

   For each sample, 1 mL of MDA extraction solution was added to 5 million
   cells, followed by ultrasonic lysis. The supernatant was collected and
   kept on ice. A working solution was prepared by mixing 300 µL of MDA
   reagent, 100 µL of the sample, and 100 µL of reagent three. The mixture
   was incubated in a 100 °C water bath for 60 min, cooled on ice, and
   centrifuged to collect the supernatant. Absorbance was measured at 532
   and 600 nm using a microplate reader. The MDA content for each group
   was calculated based on the absorbance and cell count.

Iron Ion Detection

   Cell samples were collected, and 0.2 mL of reagent one was added 1 ×
   10^6 per cell. After lysis and centrifugation, the supernatant was
   collected and kept on ice. For the assay, 80 µL of standard solution
   was added to the standard wells of a microplate, and 80 µL of the
   sample was added to the test and control wells. Then, 80 µL of reagent
   two was added to the control wells, while 80 µL of reagent three was
   added to the test and standard wells. The solutions were mixed and
   incubated at 37 °C for 10 min. OD values at 593 nm were measured using
   a microplate reader, and the iron ion content in the samples was
   calculated based on the standard curve.

BMDM and BMDC Extraction and Induction

   Bone marrow was harvested from the femurs and tibias of 6‐week‐old
   C57BL/6 mice using pre‐chilled PBS. The marrow was filtered and
   dissociated to ensure single‐cell suspensions. Cells were centrifuged,
   and the supernatant was discarded. Red blood cell lysis buffer was
   added to the cell pellet for 3 min, followed by the addition of PBS to
   stop lysis. After further centrifugation and washing steps, the cells
   were transferred to culture plates. BMDC Induction: Cells were cultured
   in RPMI‐1640 medium supplemented with 20 ng mL^−1 GM‐CSF and
   10 ng mL^−1 IL‐4 for 6 days. BMDM Induction: Cells were cultured in
   DMEM supplemented with 20 ng mL^−1 M‐CSF for 6 days. Induced BMDCs and
   BMDMs were used for subsequent experiments.

SCLC Syngeneic Tumor Model

   C57BL/6 male mice (6–8 weeks old) were subcutaneously injected with
   100 µL of PBS containing 1 × 10^6 RPM cells into the right inguinal
   region. After 7 days, mice were randomly divided into five groups:
   PRL@RBCM‐cRGD, PTX, siNC/PRL@RBCM‐cRGD, siPFKFB4/PRL@RBCM‐cRGD, and
   siPFKFB4/PRL[PTX]@RBCM‐cRGD. PTX was administered at a dose of 10
   mg kg^−1 once a week for four weeks. Tumor volume and mouse weight were
   recorded every three days. Tumor volume was calculated using the
   formula: Volume = 0.5 × Length × Width^2 . At the end of the
   experiment, blood was collected via cardiac puncture, and serum was
   extracted. Mice were euthanized using CO₂, and tissues including the
   heart, liver, spleen, lungs, kidneys, and tumors were harvested for
   subsequent analysis. Combination Therapy with aPD‐L1: Tumor‐bearing
   mice were randomized into four groups: PRL@RBCM‐cRGD + vector,
   PRL@RBCM‐cRGD + aPD‐L1, siPFKFB4/PRL[PTX]@RBCM‐cRGD + vector, and
   siPFKFB4/PRL[PTX]@RBCM‐cRGD + aPD‐L1. All animal studies were approved
   by the Ethics Committee of Zhujiang Hospital, Southern Medical
   University (Approval Number: LAEC‐2023‐223) and conducted in compliance
   with the principles of the Helsinki Declaration.

In Vivo Uptake Study

   C57BL/6 mice bearing subcutaneous RPM cell tumors in the right inguinal
   region were randomly divided into two groups. Each group received tail
   vein injections of PRL or PRL@RBCM‐cRGD. Mice were imaged at 2, 6, and
   24 h post‐injection using an IVIS Spectrum imaging system. At 24 h,
   mice were euthanized, and organs including the heart, liver, spleen,
   lungs, kidneys, and tumors were harvested to evaluate biodistribution.
   Data analysis was performed using the Living Image software.

Immunohistochemistry and immunofluorescence

   Tumor tissues from different treatment groups were dehydrated,
   embedded, and sectioned to obtain paraffin slices. Sections were
   deparaffinized, subjected to antigen retrieval, and blocked. Primary
   antibodies were added, and sections were incubated overnight at 4 °C.
   The next day, sections were washed with PBS and incubated with
   secondary antibodies at room temperature in the dark for 1 h. DAB or
   TAB reagent was added for further incubation, followed by DAPI staining
   for nuclei visualization. Slides were sealed and imaged under a
   fluorescence microscope. This study was approved by the Ethics
   Committee of Zhujiang Hospital (2024‐KY‐129‐01), Southern Medical
   University, and conducted in compliance with the Declaration of
   Helsinki.

Flow Cytometry

   Tumor samples were digested and processed to single‐cell suspensions,
   followed by red blood cell lysis. Cells were incubated with surface
   antibodies in the dark for 30 min, fixed, permeabilized, and stained
   with intracellular antibodies. The following markers were used: M1
   Macrophages: CD45⁺F4/80⁺CD86⁺, M2 Macrophages: CD45⁺F4/80⁺CD206⁺, DCs:
   CD45⁺CD11c⁺MHC‐II⁺, CD8⁺ T Cells: CD45⁺CD3⁺CD8⁺. The samples were
   analyzed using a CytoFLEX flow cytometer. Data were processed and
   visualized using FlowJo 10.8.1 software. Detailed information about the
   antibodies used is provided in the [193]Supporting Information.

Data Analysis

   All data were obtained from three independent experiments and expressed
   as mean ± standard error of the mean (SEM). Statistical analyses were
   performed using SPSS software. The following tests were employed:
   Independent Samples t‐Test, Wilcoxon Test, and One‐Way ANOVA,
   Differences were considered statistically significant if p < 0.05.
   Graphical representations were created using Origin 2021 software, and
   final figures were arranged using Adobe Illustrator 2021.

Conflict of Interest

   The authors declare no conflict of interest.

Author Contributions

   X.L., J.H., and H.Y. contributed equally to this work. This study was a
   collaborative effort of all authors. X.L. was responsible for the
   synthesis and characterization of nanoparticles, experimental
   validation, data analysis, and manuscript preparation. J.H. conducted
   the animal experiments. H.Y. performed sequencing data analysis. P.L.
   and W.Z. contributed to data analysis and verification. C.C. was
   primarily involved in the cell experiments. C.Z. managed the primary
   cell culture and induction. T.W. oversaw the overall project. B.T. was
   in charge of project management, experimental design, and manuscript
   review. J.Z. supervised the overall progress of the project and
   facilitated the acquisition of funding from the National Natural
   Science Foundation of China. All authors approved the final version of
   the manuscript for submission.

Supporting information

   Supporting Information
   [194]ADVS-12-2417374-s001.docx^ (3.9MB, docx)

Acknowledgements