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Macrophage membrane-biomimetic adhesive polycaprolactone nanocamptothecin for improving cancer-targeting efficiency and impairing metastasis



doi: 10.1016/j.bioactmat.2022.06.013.


Epub 2022 Jun 23.

Affiliations

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Kangkang Ying et al.


Bioact Mater.


2023 Feb.

Abstract

The recent remarkable success and safety of mRNA lipid nanoparticle technology for producing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines has stimulated intensive efforts to expand nanoparticle strategies to treat various diseases. Numerous synthetic nanoparticles have been developed for pharmaceutical delivery and cancer treatment. However, only a limited number of nanotherapies have enter clinical trials or are clinically approved. Systemically administered nanotherapies are likely to be sequestered by host mononuclear phagocyte system (MPS), resulting in suboptimal pharmacokinetics and insufficient drug concentrations in tumors. Bioinspired drug-delivery formulations have emerged as an alternative approach to evade the MPS and show potential to improve drug therapeutic efficacy. Here we developed a biodegradable polymer-conjugated camptothecin prodrug encapsulated in the plasma membrane of lipopolysaccharide-stimulated macrophages. Polymer conjugation revived the parent camptothecin agent (e.g., 7-ethyl-10-hydroxy-camptothecin), enabling lipid nanoparticle encapsulation. Furthermore, macrophage membrane cloaking transformed the nonadhesive lipid nanoparticles into bioadhesive nanocamptothecin, increasing the cellular uptake and tumor-tropic effects of this biomimetic therapy. When tested in a preclinical murine model of breast cancer, macrophage-camouflaged nanocamptothecin exhibited a higher level of tumor accumulation than uncoated nanoparticles. Furthermore, intravenous administration of the therapy effectively suppressed tumor growth and the metastatic burden without causing systematic toxicity. Our study describes a combinatorial strategy that uses polymeric prodrug design and cell membrane cloaking to achieve therapeutics with high efficacy and low toxicity. This approach might also be generally applicable to formulate other therapeutic candidates that are not compatible or miscible with biomimetic delivery carriers.


Keywords:

Antimetastasis; Cancer nanomedicine; Macrophage membrane; Nanocamptothecin; Polymer prodrug.

Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures



Graphical abstract


Fig. 1


Fig. 1

(a) Schematic illustration of the procedure used to prepare macrophage membrane-camouflaged polymeric nanotherapy (mSLP). The esterase-activatable SN38 prodrug (PCLn-SN38, n = 28) was synthesized and stabilized by lipid components. The SN38 lipid nanoparticles (SLP) were sequentially cloaked with LPS-stimulated M1-type macrophage membranes to increase cell adhesion and tumor cell uptake. (b) Following intravenous injection, mSLP had prolonged systemic circulation and preferentially accumulated at tumor sites. After uptake by cancer cells, mSLP effectively released the DNA topoisomerase I inhibitor SN38 in response to intracellular esterase to inhibit tumor growth and metastatic burden.


Fig. 2


Fig. 2

Preparation and characterization of macrophage membrane-camouflaged nanocamptothecin. (a) Real-time PCR analysis of the mRNA expression of M1-type macrophage markers. Transmission electron microscopy images of (b) uncoated lipid nanoparticle SLP and (c) membrane-cloaked mSLP. Insets show the size distribution of nanoparticles measured using dynamic light scattering (DLS). (d) Hydrodynamic diameter (DH), (e) polydispersity index (PDI), and (f) zeta potential of nanoparticles. (g) Protein profiles of SLP, M1-type macrophage cell membrane (CM) and mSLP analyzed using SDS–PAGE. (h) Expression of integrin α4 and integrin β1 in each sample measured using western blotting. (i) Stability assessed by measuring changes in the size and PDI in phosphate-buffered saline (PBS) or in PBS containing 10% (v/v) FBS at 37 °C. (j) In vitro drug release profiles in PBS (pH = 7.4) and PBS containing 30 U/mL porcine liver esterase (PLE). Data are presented as the means ± SD.


Fig. 3


Fig. 3

In vitro cytotoxicity of nanocamptothecin. The cytotoxicity of CPT-11, free SN38, SLP and mSLP toward (a) B16F10 and (b) 4T1 cancer cells, as determined using the CCK-8 assay. IC50 values were calculated based on the dose–response curves. (c) A schematic diagram illustrating the experimental protocol for drug withdrawal. Viability of (d) B16F10 and (e) 4T1 cancer cells after an incubation with drugs for 6 h, 12 h and 24 h, and subsequent incubation with fresh medium. Data are presented as the means ± SD. *p < 0.05, and **p < 0.01.


Fig. 4


Fig. 4

Cell apoptosis, proliferation inhibition, and cell cycle arrest induced by nanocamptothecin. Apoptosis of (a) B16F10 and (b) 4T1 cells after drug treatment was detected by staining with FITC Annexin V/PI and flow cytometry analysis (n = 3). Proliferation of (c and d) B16F10 and (e and f) 4T1 cells after exposure to drugs, as determined using the Click-iT EdU assay (n = 3). Cell cycle distribution of B16F10 (g) and 4T1 (h) cells after exposure to drugs, as determined by flow cytometric analysis (n = 3). Data are presented as the means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001.


Fig. 5


Fig. 5

Adhesion and cellular uptake of macrophage membrane-cloaked nanocamptothecin. (a) Nanoparticle adhesion to 4T1 and B16F10 cells was examined using confocal laser scanning microscopy (CLSM). The cell membrane was labeled with lectin (green), and cell nuclei were stained with Hoechst (blue). Flow cytometry analysis of DiI-labeled nanoparticle cellular uptake after 4 h of incubation at 37 °C in (b) 4T1 and (c) B16F10 cells (n = 3). (d) Intracellular distribution of DiI-labeled mSLP observed with time-lapse confocal microscopy. Endo/lysosomes were stained with LysoTracker (green), and cell nuclei were stained with Hoechst (blue). (e and f) Uptake pathway of mSLP analyzed using flow cytometry. Cells were pretreated with various inhibitors of different endocytosis pathways (n = 3). Data are presented as the means ± SD. ***p < 0.001.


Fig. 6


Fig. 6

Pharmacokinetic study and in vivo tumor targeting capacity of nanoparticles. (a) Plasma SN38 concentration-time profiles in SD rats after a single intravenous injection of CPT-11, SLP or mSLP via the tail vein. The drug dose was 10 mg/kg (SN38 equiv.). (b) Real-time in vivo NIR fluorescence images of mice bearing orthotopic 4T1 breast tumors after an intravenous injection of DiR-labeled nanoparticles. (c) Ex vivo fluorescence images of excised tumors and major organs (heart, liver, spleen, lung and kidneys) captured at 24 h post injection. BF: bright field. (d) Quantitative analysis of the average radiant efficiency in major organs (n = 4). (e and f) Images and average radiant efficiency of tumors from SLP- and mSLP-treated mice (n = 4). (g and h) Fluorescence images and intensities of tumor tissue sections visualized using fluorescence microscopy (n = 3). (i) Drug concentrations in the mouse model of orthotopic 4T1 breast cancer after a single injection of nanotherapies at 15 mg/kg SN38 equiv. dose. Drug concentrations were determined by HPLC analysis at 8, 24, and 48 h postadministration. (j) Real-time in vivo NIR fluorescence images of mice after intravenous injection of DiR-labeled nanoparticles. (I) SLP, in the non-metastatic mouse model; (II) SLP, in the spontaneous metastatic mouse model; (III) mSLP, in the non-metastatic mouse model; (IV) mSLP, in the spontaneous metastatic mouse model. (k) Ex vivo fluorescence images of major organs (liver, spleen and lung) and excised tumors, and quantitative analysis of fluorescence intensities obtained at 8 h, 24 h and 48 h post injection (n = 3). Data are presented as the means ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001.


Fig. 7


Fig. 7

In vivo antitumor activity of nanoparticles in preclinical TNBC mouse models bearing 4T1 tumors (ad) and Py8119 tumors (ei). (a) Tumor progression curves in the 4T1 orthotopic tumor-bearing mouse model after different drug treatments (n = 8). (b) Survival curves of mice from different treatment groups (n = 8). (c) Body weight changes monitored in mice from each group (n = 8). (d) H&E, Ki67 and TUNEL staining of tumor sections. (e) Tumor progression curves in the Py8119 orthotopic tumor-bearing mouse model after different drug treatments (n = 8). (f) Body weight changes monitored in mice from each group (n = 8). (g and h) Photograph and weights of excised tumors in each group at the endpoint of the study. (i) H&E, Ki67 and TUNEL staining of tumor sections. Data are presented as the means ± SD. **p < 0.01, and ***p < 0.001.


Fig. 8


Fig. 8

In vivo antimetastatic activity of bioadhesive nanocamptothecin in a 4T1 spontaneous metastasis model. (a) Representative images of major organs and lymph nodes excised from each treatment group. i) Right axillary lymph node; ii) left axillary lymph node; iii) right inguinal lymph node; iv) left inguinal lymph node. (b) Weights of lymph nodes from different groups (n = 3). ILN, inguinal lymph node; ALN, axillary lymph node. (c and d) Histological analysis of the right inguinal lymph node, liver, and lung using H&E staining. (e) Statistical histogram of spleen weights in each group (n = 3). (f) H&E staining of the spleen. Data are presented as the means ± SD. *p < 0.05 and **p < 0.01.

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