Chemically Engineered Immune Cell-Derived Microrobots and Biomimetic Nanoparticles: Emerging Biodiagnostic and Therapeutic Tools

Abstract

Over the past decades, considerable attention has been dedicated to the exploitation of diverse immune cells as therapeutic and/or diagnostic cell-based microrobots for hard-to-treat disorders. To date, a plethora of therapeutics based on alive immune cells, surface-engineered immune cells, immunocytes' cell membranes, leukocyte-derived extracellular vesicles or exosomes, and artificial immune cells have been investigated and a few have been introduced into the market. These systems take advantage of the unique characteristics and functions of immune cells, including their presence in circulating blood and various tissues, complex crosstalk properties, high affinity to different self and foreign markers, unique potential of their on-demand navigation and activity, production of a variety of chemokines/cytokines, as well as being cytotoxic in particular conditions. Here, the latest progress in the development of engineered therapeutics and diagnostics inspired by immune cells to ameliorate cancer, inflammatory conditions, autoimmune diseases, neurodegenerative disorders, cardiovascular complications, and infectious diseases is reviewed, and finally, the perspective for their clinical application is delineated.

Keywords: artificial dendritic cell and extracellular vesicle; biomimetic drug delivery; engineered immune cell; immune cell membrane; nanomedicine.

Figure 1

Light responsive NP‐loaded DC‐based cancer vaccine. a) The photosensitizer is grafted to the polymer via a conventional carbodiimide reaction. b) Positive chains of PheoA‐PEI interact with negatively charged cancer antigen, OVA, and spherical antigen containing NPs are formed. c) NPs were endocytosed and located within the endolysosomal compartments until being irradiated. After that, because of PheoA‐induced ROS production, antigens were liberated in the cytoplasm and presented to T cells. d) Fluorescent signals of antigen and lysosome were colocalized before being irradiated using a 670 nm laser source, proceeded by OVA being dispersed within the cytoplasm. e,f) Tumor volume in mice receiving each of the test or control formulations. In the photographs, I, II, III, and IV indicate the tumors of animals that received nontreated DCs, OVA‐treated DCs, DCs incubated with PheoA‐PEI–OVA without irradiation, and DCs incubated with PheoA‐PEI–OVA with irradiation, respectively. Reproduced with permission.^{[ 26 ]} Copyright 2017, American Chemical Society.

Figure 2

Dendritic cell‐mediated delivery of ND‐PG‐RGD‐DOX to glioblastoma. a) Synthesis procedures of the ND‐PG‐RGD‐DOX nanocomposite from dND‐PG‐N₃. The procedures of dND‐PG‐N₃ synthesis include 1) glycidol, 140 °C, 20 h; 2) p‐TsCl, NaOH, 0 °C and room temperature overnight; 3) NaN₃, 90 °C, overnight. Procedures for the modification of dND‐PG‐N₃ with RGD and DOX include i) bis(4‐nitrophenyl) carbonate, triethylamine, room temperature, 24 h; ii) hydrazine monohydrate, 90 °C, overnight; iii) RGD propargyl amide, copper (II) sulfate pentahydrate, sodium ascorbate, room temperature, 48 h; iv) DOX hydrochloride, pH 7, 50 °C, 24 h. Adapted with permission.^{[ 29 ]} Copyright 2014, Elsevier. b) Fluorescent imaging of mouse brain tissue sections shows the presence of cells and mDC‐delivered NanoDOX in orthotopic glioblastoma cell xenografts. Nucleus was stained with blue Hoechst 33342 fluorescent agent. Red fluorescence came from DOX or NanoDOX and green fluorescence from the CFSE‐labeled mDC. c) Immunohistological staining of caspase‐3 activity, as a marker of apoptosis, in mouse orthotopic glioblastoma cell xenografts treated differently. Caspase‐3 activity was dramatically intensified in xenografts that received NanoDOX‐mDC or NanoDOX‐mDC/mLC, but was mildly increased in xenografts that were treated with mLC or mDC/mLC. Adapted with permission.^{[ 30 ]} Copyright 2018, Elsevier.

Figure 3

a) Tunneling nanotubes between M1‐Dox and SKOV3 ovarian carcinoma cells. b,c) Red fluorescence of DOX presenting inside green tunneling nanotube connected cells were observed by confocal laser scanning microscopy (CLSM). Phalloidin (green) was used to stain F‐actin as the vital component of tunneling nanotubes, and DOX emitted intrinsic red fluorescence. d) The fluorescence images of the tumor and excised organs 24 h after the i.p. injection of d1) M1‐DiD and d2) Lipo‐DiD. Color bars show the fluorescent intensity. High accumulation of the Lipo‐DiD in the liver is observable. e,f) Final tumor weights and photographs of metastatic nodules collected on several peritoneal organs after different treatments for 28 days. Adapted with permission.^{[ 39 ]} Copyright 2018, American Chemical Society.

Figure 4

a) Schematic illustration of developing macrophage‐based microrobot with PLGA–DTX–Fe₃O₄ (left) and depiction of possible tumor targeting in vivo (right). b) Mϕs (green) that have internalized NPs (red) are distinguished by fluorescence imaging. c) Active targeting of tumor spheroid with magnetically guided Mϕs. d) Toxicity test on the murine colorectal CT‐26 cancer cell line. Reproduced with permission.^{[ 45d ]} Copyright 2016, Springer Nature.

Figure 5

a) Preparation of core–shell NR@DOX:SA nanoplatform programmed for cell‐mediated drug delivery. Efficient controlled drug release was achieved through NIR light irradiation. The TEM image of NR@SAs shows monodisperse particles. Scale bar is 200 nm. b) Immunofluorescence staining of tumor sections after treatment by the therapeutic macrophage and pristine nanoparticles in different conditions. Hoechst was administered i.v. and stained nuclei in blue color and hypoxia areas were visualized by PIMO (green) marker. Normoxia region (N) referred to the areas in which blood flow and Hoechst staining (blue) was observed without any hypoxia signal (green), and vice versa for defining hypoxia region (H). The white circle indicates the accumulation of therapeutic macrophages in the hypoxic regions. Scale bar is 100 µm. Reproduced with permission.^{[ 50 ]} Copyright 2017, Ivyspring International Publisher.

Figure 6

a–c) Schematic depiction of the artificially reprogrammed HION‐loaded Mϕs (HION@Macs) and targeting of the tumor tissue through active chemotaxis and magnet guidance. Engineered HION@Macs induce more production of inflammatory cytokines (such as NO and TNF‐α), resulting in the in situ re‐education of M2 macrophages into proinflammatory M1 phenotype for cancer therapy. d) Flow cytometry analysis of CD80 expression in pristine M1 Mϕs, M1 Mϕs cultured in M2 medium for 24 h, HION@Macs, and HION@Macs after treatment with M2 medium for 24 h. M2 medium has a negligible effect on the phenotype transition from M1 to M2 in the HION@Macs. e) Tumor volume profile of animals for 21 days. Three injections of different formulations on the 1st day, 3rd day, and 5th day (designated by a red arrow) were conducted in tumor‐bearing BALB/c mice once tumor volume reached ≈80 mm³. The asterisks indicate the difference between the HION@Macs + magnet group, the HION@Macs group, and the PBS group. ^** p < 0.01; ^*** p < 0.001. f) Representative immunofluorescence staining images for iNOS (green) and immunohistochemistry staining images for Ki‐67⁺ areas (gray dots) of tumor sections from different groups on the 7th day post‐treatment. Reproduced with permission.^{[ 54 ]} Copyright 2019, Wiley‐VCH.

Figure 7

a) Schematic illustration of M‐SMN preparation and free drug or vesicle release after MC to Mϕ switching for antimetastatic purpose. Mertansine was first attached to the legumain sensitive peptide linker. Next, the conjugation of drug‐linker to the SMA polymer resulted in the formation of SMA‐AANK‐mertansine. b) TEM image of the developed SMNs in the absence (left) and the presence (right) of legumain. c) The schematic depiction of studying the inhibitory effect of M‐SMNs on the proliferation of 4T1 cancer cells. d) The inhibitory effect was visualized by Live/Dead assays at 48 h postincubation of 4T1 cells with MCs, mertansine (0.5 µg mL⁻¹), SMNs (0.5 µg mL⁻¹ of mertansine), and M‐SMNs (1 × 10⁵ cells per well). e,f) The migration and invasion activities of metastatic 4T1 cells were measured by transwell‐mediated assays. g) Formation of metastatic nodules (pale spots) in the lungs of mice treated with saline (control), free mertansine molecules, SMNs, MCs, and M‐SMNs. Adapted with permission.^{[ 60 ]} Copyright 2017, American Chemical Society.

Figure 8

Neutrophils carrying PTX‐loaded CL for suppressing postoperative malignant glioma recurrence. a) Synthesis of the cationic lipid HG2C₁₈ for fabricating CLs. b) Schematic depiction of PTX‐CL/NEs formation and its effect on the suppression of postoperative glioma recurrence in mice. Surgical resection of a glioblastoma induces amplified inflammatory signals in the brain, which allows PTX‐CL/NEs to target brain tumors and release PTX to suppress the recurrence of glioma. SPC, Chol, and HG2C₁₈ refer to soy phosphatidylcholine, cholesterol, and 1,5‐dioctadecyl‐N‐histidyl‐l‐glutamate, respectively. c) Quantification of the PTX distribution in the brain of the surgically treated G422‐bearing mice after i.v. administration of different PTX formulations at a PTX dosage of 5 mg kg⁻¹ over time. Data are shown as mean ± s.d. of three independent experiments. ^** p < 0.01 and ^*** p < 0.001 (two‐tailed Student's t‐test). ** represents p < 0.01. Reproduced with permission.^{[ 76 ]} Copyright 2017, Springer Nature.

Figure 9

Surface‐engineered Mϕs for targeting of lung metastasis. a) Fabrication of Mϕs and their transformation to drug‐loaded vesicles in the presence of legumain in the tumor site. b) Field emission‐TEM image of the secondary drug‐containing vesicles from tumor cells treated with the drug‐loaded Exo‐like nanovesicles. c,d) The superior effect of the engineered Mϕs in reducing tumor cells’ viability and migrating ability in vitro. e) Percent of the drug released in the form of free molecules or entrapped in the secondary vesicles in the supernatant of the tumor cells treated with the drug‐loaded Exo‐like nanovesicles. f) Typical imaging and histological examination of lung tissues of the animal subjects that received each of the test or control groups (metastatic nodules are pointed out). Adapted with permission.^{[ 95b ]} Copyright 2018, American Chemical Society.

Figure 10

SN‐38‐loaded multilamellar liposomes attached to the TC's surface for targeting disseminated lymphoma tumors. a) Components of the nanoliposomes. b) The procedure for fabricating the NPs. c) Schematic of cell‐mediated delivery of SN‐38 NCs into tumors via TC functionalization. Reproduced with permission.^{[ 102 ]} Copyright 2015, American Association for the Advancement of Science.

Figure 11

a) Scheme of NG synthesis and protein release in response to TC reducing activity in the local microenvironment. TEM image of NGs prepared from IL‐15Sa is shown. Scale bar is 50 nm. b) Schematic representation of surface modification of cytokine‐NGs with anti‐CD45 Ab and PEG‐PLL to facilitate stable attachment to T cell surfaces. c) Flow cytometric analysis of primed pmel‐1 CD8+ TCs that were coupled with fluorescently labeled aCD45/IL‐15Sa NGs at the indicated cytokine levels. The NG fluorescence showed that the maximum amount of IL‐15Sa loading on 10⁶ TCs is 800 ng. d) Flow cytometric analysis of primed pmel‐1 CD8+ T cells that were conjugated with aCD45/cytokine‐containing or cytokine‐only‐containing NGs. The results demonstrated the role of aCD45 on the prolonged surface remaining of the NGs. e) Experimental scheme of animal studies. Luciferase‐expressing U‐87 MG human glioblastoma cells (1.0 × 10⁶) were injected into NSG mice (n = 5 mice per group) via s.c. route. Mice received adoptive transfer of human T cells (2.6 × 10⁶ total cells, 38% transduced with EGFR‐targeting CAR (1.0 × 10⁶ CAR‐TCs)) intravenously on day 7. Treatments included sham saline, CAR‐TCs alone, CAR‐TCs + 13.8 µg of free IL‐15Sa, or CAR‐TC coupled with aCD45/IL‐15Sa‐NGs (13.8 µg). f) In vivo bioluminescence imaging of luciferase‐expressing U‐87 MG tumor cells over time. Reproduced with permission.^{[ 98c ]} Copyright 2018, Springer Nature.

Figure 12

a) Schematic depiction demonstrating the effects of the mild NIR mediated heating of the tumor on the enhanced infiltration and activation of adoptive anti‐CSPG4 CAR‐TCs. b) Representative images of hypoxia and HIF1‐α immunofluorescence staining of the tumors before and after PTT. The blue areas are stained with DAPI for the recognition of tumor cells and green areas demonstrate hypoxia and HIF1‐α active regions (scale bar is 50 µm). Reproduced with permission.^{[ 106 ]} Copyright 2019, Wiley‐VCH. c,d) Polymeric biomatrices placed directly on pancreatic tumors can effectively deliver programmed CAR‐TCs. c) Photograph of the scaffold, bright‐field microscopy of stimulatory microspheres incorporated into it, and a schematic depiction of the composition of the microspheres. Scale bar is 70 µm. d) This series of images shows: i) biopolymeric scaffold; ii) seeding of tumor‐reactive TCs into the scaffold; iii) incision; iv) orthotopic KPC pancreatic tumor; v) implantation of the TC‐loaded scaffold; vi) wound closure; vii–ix) sustained release of tumor‐reactive T cells. e) Schematic illustration of mechanism, by which the adjuvant, microparticle, and CAR‐TC coloaded scaffold can eradicate the tumor cells. The released molecules can prime host immune cells to recognize and lyse tumor cells. Reproduced with permission.^{[ 107 ]} Copyright 2017, American Society for Clinical Investigation.

Figure 13

a) Schematic depiction of CAR‐NKCs conjugated to PTX‐loaded cMLVs. CARs were derived from the single‐chain variable fragment (scFv) of an Ab and the TC receptor signaling complex. CARs were transduced into NK92 cells, and cMLVs were conjugated to the cell surface by interaction with free thiol groups. b) Fluorescence image of CAR‐NKCs conjugated to DiD‐loaded cMLVs. CAR‐NKCs were labeled with CFSE before conjugation to DiD‐labeled cMLVs. Scale bar is 5 µm. c) Tumor growth inhibition in different groups treated with different formulations. Reproduced with permission.^{[ 113 ]} Copyright 2017, American Society of Gene and Cell Therapy. d) Scheme of the final backpack composition and layers with the release region composed of PDAC/SPS for attachment to MCs. e) Representative fluorescence images of ELIP backpacks anchored to MCs. The green fluorescence originated from phospholipids that were fluorescently labeled and embedded into the liposomes, while the red fluorescence was a result of fluorescently labeled NeutrAvidin in the attachment region (DyLight 550). Reproduced with permission.^{[ 114 ]} Copyright 2015, Wiley‐VCH. f) Schematic of CRP detection by aptamer‐conjugated PBMCs. Sulfo‐NHS‐SS‐biotin was crosslinked to PBMCs, followed by the introduction of streptavidin to attach to the biotin. Next, aptamer was conjugated to the PBMCs (Apt–PBMCs). In the blood circulation, the complex can recognize CRP and form a CRP–aptamer–PBMC complex, and then, a detectable fluorescence signal was emitted by attaching Ab coated‐beads or anti‐CRP Abs to the conjugated complex. g) TNF‐α profiling assay after treating both PBMCs and Apt–PBMCs (1 × 10⁵ cells per well) with LPS (0.2 µg mL⁻¹) for 24 h (^*** p‐value < 0.0001). h) Trypan blue assay for the counting of viable PBMCs and Apt–PBMCs (seeding density = 1.5 × 10⁵ per well) after 72 h. i) Fluorescence images of CRP capturing by Apt–PBMCs using a labeled fluorescence Ab at three different concentrations of CRP (1, 5, and 30 mg L⁻¹). Reproduced with permission.^{[ 116 ]} Copyright 2016, Springer Nature.

Figure 14

a) Pluronic P123‐lipids‐leukocyte membrane proteins liposome for breast cancer targeting and preventing lung metastasis. Leukocyte membrane proteins were used to confer biomimetic functions to the particles. b) Antitumor growth was obtained by PTX, while Pluronic P123 did not allow the lung metastasis of breast cancer. Leukocyte membrane proteins were used for targeting and biomimicry purposes. Adapted with permission.^{[ 133 ]} Copyright 2019, Royal Society of Chemistry.

Figure 15

a) Scheme of fabricating leukocyte‐mimicking immunomagnetic nanoplatform and its potential for CTCs isolation from blood samples. b) In vitro capture efficiency of Ab modified and nonmodified BIMNs toward MCF‐7 and HepG2 cells as two antigen‐positive cells as well as Jurkat and J774A.1 as two antigen‐negative cells. c) CTCs enumeration in 1.5 mL blood of eight cancer patients. All bars represent means ± SD (n = 3). Reproduced with permission.^{[ 150 ]} Copyright 2019, Wiley‐VCH.

Figure 16

a) MϕCM enveloped gold–silver nanocages intended for drug delivery and PTT of localized infection. b) Flow cytometry characterization of Mϕs pretreated with bacteria, showing high expressions of TLR2 and TLR4 in Mϕs. c) TEM image of nanocages before coating with the membrane of Mϕs pretreated with S. aureus. d) TEM image of nanocages after coating with the membrane of Mϕs pretreated with S. aureus. Scale bar is 200 nm. Inserted image scale bar is 20 nm. e) The pattern of drug release from the enveloped nanocages with and without NIR irradiation. f) Antibacterial efficiency of enveloped NPs with or without NIR irradiation (scale bar is 2 cm). g) Quantitative analysis and photographs of the bacterial colony of the infected tissues treated differently by NIR irradiation (808 nm, 1.0 W cm⁻², 5 min). GSNC, M‐GSNC, Sa‐M‐GSNC, and Ec‐M‐GSNC refer to gold–silver nanocage, MϕCM enveloped gold–silver nanocages, Mϕ membrane enveloped gold–silver nanocages where the membrane was pretreated with S. aureus, and MϕCM enveloped gold–silver nanocages where the membrane was pretreated with E. coli, respectively. Adapted with permission.^{[ 149 ]} Copyright 2018, Wiley‐VCH.

Figure 17

a) Illustration of the preparation procedure of the membrane‐coated nanoparticles for targeted and on‐demand drug release. b) Penetration efficiency of PPC8@Ma and cskc‐PPiP@Ma into tumor spheroids with 2 h of incubation in culturing media with two different pH values of 7.4 and 6.5. Scale bar is 100 µm and the z‐axis depth is 20 µm. c) Cumulative drug‐release profile of PPiP/PTX@Ma at various pH values of 7.4, 6.5, and 5, mimicking representatives of the physiologic, tumor microenvironment, and endosomal pH, respectively. d) Quantification of PTX concentration in tumor tissue and different organs of mice after receiving commercial Taxol or different formulations. e) 3D reconstruction of fluorescence signal accumulation in the mouse body 48 h after treatment with cskc‐PPiP@Ma. Reproduced with permission.^{[ 151 ]} Copyright 2018, American Chemical Society.

Figure 18

a) Scheme of the fabrication procedure of leutusomes for enhanced solid tumor targeting. b) Verification and characterizations of fusing Mϕ and tumor cell membranes into leutusome. Visualization of extracted dual fluorescent‐labeled composite cells membrane before and after extrusion was performed using confocal microscopy and TEM imaging. DiD was used for the labeling of leukocyte membranes (green fluorescence) and DiO was used for the labeling of tumor cell membranes (red fluorescence). c) Antitumor effect of the different formulations. PTXL refers to PTX‐loaded liposomes without any cell membrane fusion. d) Tumor tissue apoptosis induction after treatment with different formulations. Green fluorescence shows apoptotic regions. Reproduced with permission.^{[ 140 ]} Copyright 2018, American Chemical Society.

Figure 19

a) Schematical represention of the process of HV generation using the cell membrane of HL 60 NEs and cryo‐TEM of the final product. b) Western blot of HL 60 cells and their HVs to assess the expression of integrin β2 and actin. Integrin β2 on HVs promotes their binding to ICAM‐1 overexpressed HUVECs and their internalization. c) Quantification of DNA and proteins in each step of centrifugation. The pellet after centrifugation at 2000 × g contained nucleus. The supernatant of the samples after centrifugation at 100 000 × g contained intracellular proteins and released DNA, while its pellet was the final HVs that had very low amount of DNA and protein. d) Fluorescence confocal images of HUVECs after incubation with Dil‐fluorescently labeled HVs or EVs. HUVECs were treated with 100 ng mL⁻¹ of TNF‐α to increase the expression of ICAM‐1. HVs could attach and penetrate into the cells more effectively than EVs due to the surface integrin β2 on the HVs. e) HV‐TPCA‐1 could show the highest attenuation of vascular inflammation in vivo, confirmed by quantification of the TNF‐α concentration in BALF 10 h after i.v. injection of Hank's balanced salt solution (HBSS), TPCA‐1‐loaded EVs (EV‐TPCA‐1), and HV‐TPCA‐1 in mice 3 h after LPS challenge (10 mg kg⁻¹). The administered doses of TPCA‐1 were 0.33 and 1 mg kg⁻¹. Reproduced with permission.^{[ 159 ]} Copyright 2016, Elsevier.

Figure 20

a) Schematic depiction of the fabrication, biological mechanism, and different biomedical applications of the “super NEs,” prepared by modifying the surface of the GOx/CPO‐embedded ZIF‐8 NPs with the natural NE membrane. b) Confocal microscopy analysis of the lung tissues after i.v. injection of FITC‐labeled GCZ and GCZM. Mice were intravenously injected with 4t1 cells before the administration of the NPs. c) Schematic demonstration of the establishment of early lung metastasis model and therapeutic procedure in BALB/c mice. d) The images of H&E stained lung tissues after the treatment of mice with different materials at day 16 post‐tumor inoculation. The black arrows show the metastatic nodules. Scale bar is 2 mm. e) Schematic demonstration of the timeline for the establishment of S. aureus‐infected mouse model and treatment schedule. f) Representative digital images of S. aureus‐infected mice at day 7 postinjection of the bacteria. Reproduced with permission.^{[ 7 ]} Copyright 2019, Wiley‐VCH.

Figure 21

a) Schematic illustration of TNPs fabricated for reducing HIV infectivity. The cell membranes of natural CD4⁺ TCs were wrapped around polymeric cores, while preserving key coreceptors for viral targeting. By mimicking the surface antigen profile of TCs, TNPs can bind to viruses and block viral entry into the target alive cells. b) TEM images of TNPs. c) Analysis of CD4 receptor, and CCR5 and CXCR4 coreceptors related to HIV binding in TCs, T vesicle, and TNP using Western blot assay. d) Measurement of the fluorescence intensity of TNPs (100 µL, 0.5 mg mL⁻¹ protein concentration) or TCs (100 µL, ≈2.5 × 10⁶ cells) comprising an identical amount of membrane content after staining with fluorescein‐isothiocyanate‐labeled anti‐CCR5 Ab. Binding capacity and specificity of TNPs against HIV envelope glycoprotein e) gp120_IIIB and f) gp120_BaL. Inhibition of infection by g) HIV_NL4‐3 and h) HIV_Bal on PBMCs, in response to various concentrations of TNP. Reproduced with permission.^{[ 166 ]} Copyright 2018, Wiley‐VCH.

Figure 22

a) Schematic demonstration of NK cell‐membrane‐cloaked NPs for combined PDT and immunotherapy. NK cell‐membranes could elicit proinflammatory M1 Mϕs polarization in tumor for triggering antitumor immunotherapy. Infiltration of effector TCs (CD4⁺ T cells and CD8⁺ T cells) in tumors could efficiently inhibit both primary and abscopal tumors. b) TEM image of the NK‐NPs. c) Gene expression associated with the M1 phenotype in vitro following various treatments. d) In vitro level of proinflammatory IL‐12 cytokine in THP‐1 cells treated with human NK‐NPs, human NK cell membranes (NKCMs), and T‐NPs. e) The antitumor response following various treatments in vivo in abscopal tumors in terms of the tumor volume. f,g) Animal survival and body weight of animals in different groups during the in vivo study. Reproduced with permission.^{[ 172 ]} Copyright 2018, American Chemical Society.

Figure 23

a) Schematic illustration of preparing MV_B16‐DC. b) Flow cytometric analysis of the phenotypes of MV_B16, MV_DC, and MV_B16‐DC. c) Flow cytometry analysis of CD 86 expression in CD11c⁺ cells after incubating BMDCs at different conditions. d) Quantitative analysis of CD11c⁺CD86⁺ cells by flow cytometry (n = 3, *p < 0.05). e) Melanoma tumor volume after administrating various formulations (n = 6, *p < 0.05). f) Tumor weight at day 19 after treatment with different formulations (n = 6, *p < 0.05). g) Tumor inhibitory rate of melanoma (n = 6, *p < 0.05). h) Immunofluorescence staining of tumor tissues. Nucleus of cancer cells, CD8⁺ TCs, and CRT⁺ cells for ICD recognition were stained with blue DAPI, red anti‐CD8 Ab, and green anti‐CRT Ab, respectively. Reproduced with permission.^{[ 190 ]} Copyright 2017, Wiley‐VCH.

Figure 24

a) Schematic demonstration of the concept behind the study of tacrolimus‐loaded PLGA NPs cloaked with Mϕ‐derived ECVs to target rheumatoid arthritis. b) Comparing the surface protein expression of MMV and its mother Mϕ membrane (m‐membrane). c) TEM image of the MNPs. d) Protein analysis of m‐membrane, MMV, and MNP. e) Arthritis index in different tested groups over 14 days of treatment; n = 5, *p < 0.05. f) Macroscopic swelling and micro‐CT images of the arthritic paw in different groups. Reproduced with permission.^{[ 214 ]} Copyright 2018, American Chemical Society.

Figure 25

a,b) Size distribution and morphological characterization of M1‐Exos and M2‐Exos. c) Markers of Exos derived from M1 and M2 Mϕs. d) Evaluation of cell apoptosis by flow cytometry at 24 h in different groups. e–h) Body weights, photographic images of tumor size, tumor volumes, and survival rates in each group. PTX‐loaded Exos derived from M1 Mϕs have superior antitumor efficiency in breast cancer mouse models. i) The expression level of caspase‐3/β‐actin. j) The quantitative analysis of the apoptosis in the tumor tissues. Values were expressed as mean ± SD (n = 6; *p < 0.05, ^** p < 0.01, and ^*** p < 0.001 vs the control group). Reproduced with permission.^{[ 215 ]} Copyright 2019, Ivyspring International Publisher.

Figure 26

a) Size distribution and morphology of the M2‐EXOs. b) Western blot analysis showed the expression of exosome markers, including CD9, TSG101, and CD63. c) Rate of neurons’ survival being treated with M2‐EXOs after ischemia. d) Confocal imaging demonstrated the uptake of PKH‐26‐labeled exosomes (red) by neurons (green) in vitro. Scale bar is 50 µm. e) Cresyl violet staining of brain sections after 3 days of treatment with PBS and M2‐Exo post‐transient middle cerebral artery occlusion. The infarcted area is shown by dashed line. f) Bar graph of infarct volume normalized to the normal side, confirming that microglial cell‐derived Exos with M2 phenotype can promote neurons’ survival after stroke. g) Confocal image of brain tissue, demonstrating the uptake of PKH‐labeled exosomes (red) by MAP‐2+ neurons. Scale bar = 10 µm. Reproduced with permission.^{[ 220 ]} Copyright 2019, Ivyspring International Publisher.

Figure 27

Schematic illustration of the fabrication of hybrid Exo–liposome exosome through a) isolation of small EVs from J774A.1 and b) hybridization of immune cell‐derived small EVs with synthetic liposome using membrane extrusion. c) TEM images of liposomes, ECV, and hybrid Exos (HE). d) Cellular internalization of HE and liposomes in mouse Mϕs, breast tumor cells, mouse osteosarcoma cells, and normal fibroblasts. Internalization was quantified via confocal microscopy according to corrected total cell fluorescence (CTCF). Reproduced with permission.^{[ 195a ]} Copyright 2019, Elsevier.

Figure 28

a) Schematic depiction of preparing cell‐templated aAPC using red blood cells or HeLA cells. Cellular molded silica particles were further coated with lipid 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) that possesses a fluid‐like state at physiological temperatures or 1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC) that has a gel‐like state at physiological temperatures. The particles were surface conjugated with anti‐CD3ε and anti‐CD28 Abs through biotin–streptavidin reaction. b) SEM images of HeLa molded silica microparticles. Reproduced with permission.^{[ 234 ]} Copyright 2018, Wiley‐VCH.

Figure 29

a) The process of APC‐ms formation from the MSRs. b) For polyclonal T cell expansion, αCD3 and αCD28 were attached on the surface of the SLB‐MSR particles (left), while for antigen‐specific T‐cell expansion, pMHC and αCD28 were attached (right). c) Summary of APC‐ms formulations used for the TC expansion studies shown in panels (d) and (e) of the figure. d) Polyclonal expansion of primary mouse TCs that were either untreated (mock), or cultured with Dynabeads or APC‐ms. The effect of Dynabeads of varying doses (D1, D2, D3), or APC‐ms of varying formulations (A1, A2, A3, A4) were tested on TC expansion at various time points (day 7 and day 13 of the cotreatment). As for Dynabeads, the tested bead‐to‐cell ratios were 1:1 (D1), 5:1 (D2), and 25:1 (D3). e) Antigen‐specific expansion of primary mouse CD8⁺ TCs that were either untreated (mock), or cultured with various APC‐ms formulations (A3, A4, A5, A6; see panel (c) of the figure). Reproduced with permission.^{[ 235 ]} Copyright 2018, Springer Nature.

Figure 30

a) Scheme of preparing RBC‐based aAPC modified with pMHC‐I, aCD28, and IL‐2, as well as their role in activating CD8+ T cells. b) Confocal images of RBC‐based aAPCs labeled with fluorescent probes. PE green fluorescence indicated anti‐OVA257–264 (SIINFEKL) Ab labeled pMHC‐I and Cy5.5 red fluorescence showed aCD28. The scale bar is 5 µm. c,d) Quantification of IFN‐γ and TNF‐α release from the untreated T cells or T cells treated with free pMHC‐I and aCD28 (pMHC‐I&aCD28), T cells treated with RBC‐aCD28 (R‐aCD28), T cells treated with RBC‐pMHC‐I (R‐pMHC‐I), T cells treated with R‐aAPC without IL‐2 (R‐aAPC), T cells treated with R‐aAPC plus free IL‐2 (R‐aAPC + fIL‐2), and T cells treated with IL‐2 conjugated R‐aAPC (R‐aAPC‐IL‐2). Error bars were based on standard deviation (SD) of triplicate samples and p values represent *p < 0.05, ^** p < 0.01, ^*** p < 0.001, and ^**** p < 0.0001. Reproduced with permission.^{[ 249 ]} Copyright 2017, Wiley‐VCH.

Figure 31

a) Schematic illustration of the synthesis of alginate‐based microparticles. b) Microfluidic‐based production of alginate microparticles; scale bar: 100 µm. c) Optical image of the alginate microparticles. d) Hydration of preformed POPC lipid film with alginate particles could create lipid‐coated alginate microparticles to mimic cells. e) Comparing the size of microparticles at different flow rates (upper panels) with natural naïve and active T cells (lower panels). Gaussian distributions are plotted to elucidate the average sizes and standard deviations (n > 50). Insets: Fluorescent confocal images of lipid‐coated alginate microparticle, in which DiD red fluorescence dye was used for phospholipid coating. f) Maleimide‐thiol chemistry was used to functionalize DSPE‐PEG2000‐modified phospholipid shell on the surface of the particles with TCR and CD4. g) Fluorescent confocal images of microparticles, showing i) TCR, ii) CD4, iii) encapsulated IL‐2, and iv) the overlay image; scale bar is 15 µm. h) Percentage of cells that fall in the category of effector cells (CD25⁺, CD127⁻), obtained by flow cytometric analysis when the cells were cocultured with microparticles at different time points. i) Percentage of effector cells (CD44⁺ CD62L⁻), obtained by flow cytometric analysis when the cells were cocultured with microparticles at different time points. Adapted with permission.^{[ 255 ]} Copyright 2018, Wiley‐VCH.