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Review
. 2022 Apr 26;16(4):5184-5232.
doi: 10.1021/acsnano.2c01252. Epub 2022 Mar 29.

Multifunctional Lipid Bilayer Nanocarriers for Cancer Immunotherapy in Heterogeneous Tumor Microenvironments, Combining Immunogenic Cell Death Stimuli with Immune Modulatory Drugs

André E Nel  1   2   3 , Kuo-Ching Mei  1   2 , Yu-Pei Liao  1   2 , Xiangsheng Liu  1   2
Affiliations
Review

Multifunctional Lipid Bilayer Nanocarriers for Cancer Immunotherapy in Heterogeneous Tumor Microenvironments, Combining Immunogenic Cell Death Stimuli with Immune Modulatory Drugs

André E Nel et al. ACS Nano. .

Abstract

In addition to the contribution of cancer cells, the solid tumor microenvironment (TME) has a critical role in determining tumor expansion, antitumor immunity, and the response to immunotherapy. Understanding the details of the complex interplay between cancer cells and components of the TME provides an unprecedented opportunity to explore combination therapy for intervening in the immune landscape to improve immunotherapy outcome. One approach is the introduction of multifunctional nanocarriers, capable of delivering drug combinations that provide immunogenic stimuli for improvement of tumor antigen presentation, contemporaneous with the delivery of coformulated drug or synthetic molecules that provide immune danger signals or interfere in immune-escape, immune-suppressive, and T-cell exclusion pathways. This forward-looking review will discuss the use of lipid-bilayer-encapsulated liposomes and mesoporous silica nanoparticles for combination immunotherapy of the heterogeneous immune landscapes in pancreatic ductal adenocarcinoma and triple-negative breast cancer. We describe how the combination of remote drug loading and lipid bilayer encapsulation is used for the synthesis of synergistic drug combinations that induce immunogenic cell death, interfere in the PD-1/PD-L1 axis, inhibit the indoleamine-pyrrole 2,3-dioxygenase (IDO-1) immune metabolic pathway, restore spatial access to activated T-cells to the cancer site, or reduce the impact of immunosuppressive stromal components. We show how an integration of current knowledge and future discovery can be used for a rational approach to nanoenabled cancer immunotherapy.

Keywords: combination therapy; immune escape; immune landscapes; immune suppression; immunogenic cell death; liposomes; nanocarrier; pancreas cancer; silicasomes; spatial distribution; triple-negative breast cancer.

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Conflict of interest statement

Competing Financial Interests

Andre E. Nel is co-founder and equity holder in Westwood Biosciences Inc. and NAMMI Therapeutics. Nel also serves on the Board for Westwood Biosciences Inc. The remaining authors declare no conflicts of interest.

Figures

Figure 1.
Figure 1.. The importance of heterogeneous tumor immune microenvironments (TIMES) for immunotherapy.
In in spite of the advances by immune checkpoint blocking antibodies for cancer immunotherapy, only 20–30% of patients with responsive cancers mount a robust antitumor immune response, provided that they exhibit an inflamed tumor microenvironment with CTL infiltration. To improve the response rate for these cancers and add to increase the overall number of additional cancers that can be successfully treated with checkpoint blocking antibodies, a number of approaches exist to convert “cold” tumors “hot”, including endogenous and exogenous vaccination approaches. Even when successful at improving CTL recruitment, these attempts may not be enough to achieve cytotoxic killing because of: (i) the immune suppressive effects of the tumor stroma; (ii) recruitment of CD8+ T-cells that are especially excluded from contacting PDAC or TNBC cancer cells; (iii) recruitment of CD8+ T-cells, which are put under constraint by ligation of checkpoint receptors on the immune metabolic effect of the IDO-1 pathway. Thus, in addition to inflamed (“hot”) and immune-depleted (“cold”, “immune desert” or “ignored”) TIMES at the far ends of the spectrum, intermediary categories such as “immune excluded”, “immune suppressed” and “immune escape” landscapes need to be considered for TNBC and PDAC immunotherapy. This requires customized design of treatment combinations to address the challenges in each landscape. Abbreviations: Treg = FoxP3+ regulatory T-cells; MDSC = myeloid derived suppressor cells; TAM = tumor-associated macrophages; IDO-1 = Indoleamine-pyrrole 2,3-dioxygenase.
Figure 2.
Figure 2.. High-dimensional immune-profiling and multiplex immunohistochemistry (mIHC) analysis of cancer landscapes.
Panel A: mIHC analysis, single cell (sc) transcriptomics (e.g., single-cell RNA-Seq) and cytometry by time of flight (CyTOF) platforms are replacing conventional tools in discovery for understanding complex and heterogeneous tumor microenvironments, including by introducing immune response biomarkers that can be used for chemoimmunotherapy.– ATAC = assay for transposase-accessible chromatin. Reprinted with permission from ref under a Creative Commons Attribution License 4.0 (CC BY). Copyright 2020 The Authors. Panel B: While conventional IHC allows detection of cellular antigens in tissue sections through the employment of enzyme-labeled or fluorochrome-labeled antibodies to identify diverse cell types and spatial location, no more than 4 markers can be used simultaneously as a result of the constraints of chromogenic or fluorescent spectra overlap. However, advancements in dye-cycling techniques, where staining, imaging and dye inactivation are done repeatedly, have enabled the detection of multiple different antigens on the same tissue sample by mIHC analysis.
Figure 3.
Figure 3.. mIHC images of heterogeneous human PDAC immune landscapes.
Utilizing tyramide-based signal amplification, Carstens et al. examined 8 distinct markers (anti-smooth muscle actin, collagen-I, cytokeratin 8, CD3, CD8, CD4 and Foxp3) to obtain spectrally mixed and unmixed images of the heterogeneous cell populations and their spatial distribution in paraffin-embedded tumor samples from 132 PDAC patients. Panel A: Spectrally mixed image of the cell phenotype map identifying the cell populations defined by the individual markers of the multiplex stain, overlayed on the raw image. Legend: Summary of each defined cell phenotype, color code and associated markers. The scale bar equals 100 nm. Panel B: spectrally mixed (upper panel) and unmixed (lower panel) images three patients (A, B and C) with differing levels of CTL infiltration - patient A showing low infiltration, patient B medium infiltration and patient C high infiltration. The unmixed phenotype map depicts the cytokeratin positive cancer cells (green) and CTLs (red) in the tumor sites. Panel A-B adapted with permission from ref under a Creative Commons Attribution License 4.0 (CC BY). Copyright 2017 The Authors.
Figure 4.
Figure 4.. Utility of the Kras pancreatic cancer (KPC mouse model) for developing combination immunotherapy.
Panel A: The KPC genetic mouse model of pancreas cancer (Pdx1-cre/LSL-Kras G12D/p53R172H) has been widely used because of its fidelity to human PDAC, including activating Kras(G12D) mutations and loss of Trp53, associated desmoplasia, and inflammation., , The spontaneous model has been instrumental in developing a number of PDAC immunotherapy approaches that are being applied in human studies, even though differences exist for KPC vs. human PDAC immune landscapes, particularly the occurrence of an immune-rich subset in humans vs. the myeloid-dominant TIME of KPC. Noteworthy, the Kras oncogene contributes to immune suppression and immune evasion in this animal model. Due to the logistical constraints to breed a sufficient number of animals for accommodating all the treatment combinations that can be studied in one experiment, we developed an orthotopic implant model in immunocompetent B6/129 mice to perform our studies. The orthotopic implant procedure involves minor surgery for injecting 2 × 106 KPC-luc cells in the tail of the pancreas (left panel). The autopsy and bioluminescence imaging reveal primary tumor growth after 1 to 2 weeks, followed by tumor metastases after 3 to 5 weeks. Macro-metastases are marked by arrows. However, in spite of the utility of the orthotopic KPC model, it is important to note that these tumors lack an autochthonous stroma or the extensive desmoplasia seen human tumors or the spontaneous GEM. Nonetheless, the orthotopic model has proven of considerable benefit in studying chemo-immunotherapy, as we will demonstrate in later sections. Adapted with permission from ref . Copyright 2020 Elsevier (upper panel A). Reprinted with permission from ref . Copyright 2017 American Society for Clinical Investigation (lower panel A). Panel B: Spectrally unmixed mIHC image obtained from a mouse PDAC tumor, stained with tumor stroma biomarkers, as shown in the figure legend. Adapted with permission from ref under a Creative Commons Attribution License 4.0 (CC BY). Copyright 2017 The Authors.
Figure 5.
Figure 5.. Therapy-naive TNBC tumors are classified into subtypes on the basis of distinct spatial localization of CD8+ T cells.
Panel A: Representative images of CD8+ T-cell staining in the vicinity of tumor margins (top panels, dotted lines) and tumor cores (bottom panels), collected from 38 human samples. Scale bars: 100 μm. Panel B: Quantification of CD8+ T cell densities at the tumor margins (marCD8) and in the tumor cores (corCD8). Data represent the mean ± SEM. Tumor phenotypes: ID = immune desert; MR = margin-restricted; SR = stromal-restricted; FI = fully inflamed. Panel A-B reprinted in part with permission from ref . Copyright 2019 American Society for Clinical Investigation.
Figure 6.
Figure 6.. CD8+ T-cells spatial distribution landscapes in 4T1, EMT6 and Py8119 TNBC animal models.
4T1 (Balb/c), EMT6 (Balb/c) and Py8119 (C57BL/6) EC cells were orthotopically implanted in mouse mammary pads on day 0. When the tumors reached 100–150 mm3, animals were IV injected on days 8, 11 and 14 with DOX-NP (5mg/kg; Avanti Polar Lipids) or left untreated (UT). Tumors were collected on day 21 and analyzed by conventional IHC staining (panel A) or multiplex IHC (mIHC) staining (Panel B). Quantitative analysis of CD8+ cells in tumor cores and margins during conventional IHC was performed, using Aperio ImageScope software. For mIHC analysis, tumor sections were stained with primary antibodies: CD8, α-SMA and Ki-67. Quantitative analysis of CD8+ numbers in cores and margins was performed using Akoya InForm Image Analysis software. Doxorubicin treatment induced increased CD8+ T-cell recruitment in all tumor types with both staining methods. Importantly, newly recruited CD8 T-cells tended to be margin- or stroma-restricted in EMT6 and Py8119 tumors, while CTL distribution in 4T1 was across the entire landscape in most tumors with stromal restriction in 30%. The same T-cell distribution was seen with mIHC, where α-SMA staining intensity in the stromal cores, followed the order EMT6 > Py8119 > EMT6. Data are expressed as mean ± SD, n = 6.
Figure 6.
Figure 6.. CD8+ T-cells spatial distribution landscapes in 4T1, EMT6 and Py8119 TNBC animal models.
4T1 (Balb/c), EMT6 (Balb/c) and Py8119 (C57BL/6) EC cells were orthotopically implanted in mouse mammary pads on day 0. When the tumors reached 100–150 mm3, animals were IV injected on days 8, 11 and 14 with DOX-NP (5mg/kg; Avanti Polar Lipids) or left untreated (UT). Tumors were collected on day 21 and analyzed by conventional IHC staining (panel A) or multiplex IHC (mIHC) staining (Panel B). Quantitative analysis of CD8+ cells in tumor cores and margins during conventional IHC was performed, using Aperio ImageScope software. For mIHC analysis, tumor sections were stained with primary antibodies: CD8, α-SMA and Ki-67. Quantitative analysis of CD8+ numbers in cores and margins was performed using Akoya InForm Image Analysis software. Doxorubicin treatment induced increased CD8+ T-cell recruitment in all tumor types with both staining methods. Importantly, newly recruited CD8 T-cells tended to be margin- or stroma-restricted in EMT6 and Py8119 tumors, while CTL distribution in 4T1 was across the entire landscape in most tumors with stromal restriction in 30%. The same T-cell distribution was seen with mIHC, where α-SMA staining intensity in the stromal cores, followed the order EMT6 > Py8119 > EMT6. Data are expressed as mean ± SD, n = 6.
Figure 7.
Figure 7.. Use of LB-coated nanocarriers to deliver drug combinations.
Panel A: Our basic approach to drug-co-formulation in liposomes and silicasomes is to use the hydrophilic interior for remote loading of amphiphilic drugs, while employing the lipophilic environment in the LB to incorporate lipid moieties and prodrugs. The lipid moieties are comprised of natural or synthetic lipid molecules with immune stimulatory effects (Table 4), while prodrugs are prepared by conjugating agents that interfere with immune escape or immune suppressive pathways to a series of lipid molecules (Figures 14–17). Panel B: Drug remote loading is accomplished by using ammonium sulfate, sucralose octasulfate and citrate for generating proton gradients, which allow amphipathic weak-basic molecules (see examples below the schematic) to cross the LB for protonation inside the silicasome pores or lysosomal interior. The protonated drug molecule complexes to the cationic group of the trapping agent to yield a drug precipitate, which regulates drug release in the TME in cancer cells. Adapted with permission from ref . Copyright 2021 Elsevier.
Figure 8.
Figure 8.. Upscale production of Irinotecan-silicasomes for effective and safe treatment of PDAC.
Panel A: We have developed upscale production of an MSNP carrier, where the LB is used for Irinotecan (IRIN) remote loading, following encapsulation of sucralose octasulfate (TEA8SOS) in the particle pores.–, – Large batch production was made possible by using ethanol precipitation for LB coating instead of sonicating a biofilm, which has limitations for coating large particle batches. The upscale flow-through sonication procedure involves the direct introduction of an aqueous suspension of MSNP into a concentrated, ethanol suspended lipid solution, followed by controlled energy input in a flow cell sonication device. The coating mechanism is assembly of the suspended lipid monomers onto on the particle surfaces upon introduction into the aqueous environment. This approach is advantageous from the perspective that there is complete and rapid surface coating by the LB (cryoEM visualization) and avoidance of potentially toxic chloroform use. This approach allows the application of LB coating to 120 g MSNP batch sizes. The picture displays the average size dimensions and physicochemical characteristics, including IRIN loading capacity of ~40%. Adapted from ref . Copyright 2019 American Chemical Society. Panel B: Improved IRIN delivery and treatment efficacy in an orthotopic KPC model, using a silicasome vs. a liposome. The inserted table shows that the increased stability of the supported lipid bilayer improve carrier stability, circulatory half-life, leakiness and drug delivery atthe KPC tumor site, compared to a liposomal equivalent. This includes facilitated drug loading as a result of van der Waal’s forces, hydrogen bonding and electrostatic interactions with the wall of the silicasome pores. Improved drug delivery was accompanied by increased tumor cell killing at the primary and metastatic sites, as shown in the lower panel. Adapted from ref . Copyright 2016 American Chemical Society. Panel C: The silicasome carrier does not induce the bone marrow cytopenia, intestinal villi blunting and liver toxicity seen with free or a liposome encapsulated Irinotecan. Similar efficacy and safety have also been demonstrated in colon cancer models. Adapted from ref . Copyright 2016 American Chemical Society. Panel D: Silicasome uptake into the KPC tumor matrix and cancer cells was improved by co-administration of the cyclic iRGD peptide, which promotes transcytosis. Silicasomes were synthesized with an imageable gold nanoparticle core, followed by IV administration of the particles at 50 mg/kg in animals bearing orthotopic KPC tumors. Tumor tissues were collected for electron microscopy viewing after 24 hours. D-1 shows conventional and pseudocolor TEM images, demonstrating intravesicular particle transport across the tumor blood vessel wall. The vesicle numbers increased in animals receiving either separate injections or injection of the iRGD-conjugated nanocarrier. Adapted with permission from ref . Copyright 2017 American Society for Clinical Investigation. D-2 shows TEM images that demonstrate endothelial vesicles and particle localization in the tumor stroma or localized inside cancer cells. Adapted with permission from ref . Copyright 2017 American Society for Clinical Investigation. D-3 is a schematic to show the working mechanism of iRGD -induced transcytosis, which involves cyclic peptide binding to overexpressed integrins at the tumor site, peptide cleavage, and release of a CendR motif that activates the tyrosine protein kinase receptor, neurophilin-1. Adapted with permission from ref . Copyright 2018 Elsevier. This transcytosis mechanism is likely identical to the vesiculovascular organ, delineated by Nagy and Dvorek et al., who performed extensive EM analysis of multiple cancer types in humans (D-4).– Reprinted with permission from ref under the terms of the Creative Commons Attribution 2.0 License. Copyright 2008 The Authors.
Figure 8.
Figure 8.. Upscale production of Irinotecan-silicasomes for effective and safe treatment of PDAC.
Panel A: We have developed upscale production of an MSNP carrier, where the LB is used for Irinotecan (IRIN) remote loading, following encapsulation of sucralose octasulfate (TEA8SOS) in the particle pores.–, – Large batch production was made possible by using ethanol precipitation for LB coating instead of sonicating a biofilm, which has limitations for coating large particle batches. The upscale flow-through sonication procedure involves the direct introduction of an aqueous suspension of MSNP into a concentrated, ethanol suspended lipid solution, followed by controlled energy input in a flow cell sonication device. The coating mechanism is assembly of the suspended lipid monomers onto on the particle surfaces upon introduction into the aqueous environment. This approach is advantageous from the perspective that there is complete and rapid surface coating by the LB (cryoEM visualization) and avoidance of potentially toxic chloroform use. This approach allows the application of LB coating to 120 g MSNP batch sizes. The picture displays the average size dimensions and physicochemical characteristics, including IRIN loading capacity of ~40%. Adapted from ref . Copyright 2019 American Chemical Society. Panel B: Improved IRIN delivery and treatment efficacy in an orthotopic KPC model, using a silicasome vs. a liposome. The inserted table shows that the increased stability of the supported lipid bilayer improve carrier stability, circulatory half-life, leakiness and drug delivery atthe KPC tumor site, compared to a liposomal equivalent. This includes facilitated drug loading as a result of van der Waal’s forces, hydrogen bonding and electrostatic interactions with the wall of the silicasome pores. Improved drug delivery was accompanied by increased tumor cell killing at the primary and metastatic sites, as shown in the lower panel. Adapted from ref . Copyright 2016 American Chemical Society. Panel C: The silicasome carrier does not induce the bone marrow cytopenia, intestinal villi blunting and liver toxicity seen with free or a liposome encapsulated Irinotecan. Similar efficacy and safety have also been demonstrated in colon cancer models. Adapted from ref . Copyright 2016 American Chemical Society. Panel D: Silicasome uptake into the KPC tumor matrix and cancer cells was improved by co-administration of the cyclic iRGD peptide, which promotes transcytosis. Silicasomes were synthesized with an imageable gold nanoparticle core, followed by IV administration of the particles at 50 mg/kg in animals bearing orthotopic KPC tumors. Tumor tissues were collected for electron microscopy viewing after 24 hours. D-1 shows conventional and pseudocolor TEM images, demonstrating intravesicular particle transport across the tumor blood vessel wall. The vesicle numbers increased in animals receiving either separate injections or injection of the iRGD-conjugated nanocarrier. Adapted with permission from ref . Copyright 2017 American Society for Clinical Investigation. D-2 shows TEM images that demonstrate endothelial vesicles and particle localization in the tumor stroma or localized inside cancer cells. Adapted with permission from ref . Copyright 2017 American Society for Clinical Investigation. D-3 is a schematic to show the working mechanism of iRGD -induced transcytosis, which involves cyclic peptide binding to overexpressed integrins at the tumor site, peptide cleavage, and release of a CendR motif that activates the tyrosine protein kinase receptor, neurophilin-1. Adapted with permission from ref . Copyright 2018 Elsevier. This transcytosis mechanism is likely identical to the vesiculovascular organ, delineated by Nagy and Dvorek et al., who performed extensive EM analysis of multiple cancer types in humans (D-4).– Reprinted with permission from ref under the terms of the Creative Commons Attribution 2.0 License. Copyright 2008 The Authors.
Figure 8.
Figure 8.. Upscale production of Irinotecan-silicasomes for effective and safe treatment of PDAC.
Panel A: We have developed upscale production of an MSNP carrier, where the LB is used for Irinotecan (IRIN) remote loading, following encapsulation of sucralose octasulfate (TEA8SOS) in the particle pores.–, – Large batch production was made possible by using ethanol precipitation for LB coating instead of sonicating a biofilm, which has limitations for coating large particle batches. The upscale flow-through sonication procedure involves the direct introduction of an aqueous suspension of MSNP into a concentrated, ethanol suspended lipid solution, followed by controlled energy input in a flow cell sonication device. The coating mechanism is assembly of the suspended lipid monomers onto on the particle surfaces upon introduction into the aqueous environment. This approach is advantageous from the perspective that there is complete and rapid surface coating by the LB (cryoEM visualization) and avoidance of potentially toxic chloroform use. This approach allows the application of LB coating to 120 g MSNP batch sizes. The picture displays the average size dimensions and physicochemical characteristics, including IRIN loading capacity of ~40%. Adapted from ref . Copyright 2019 American Chemical Society. Panel B: Improved IRIN delivery and treatment efficacy in an orthotopic KPC model, using a silicasome vs. a liposome. The inserted table shows that the increased stability of the supported lipid bilayer improve carrier stability, circulatory half-life, leakiness and drug delivery atthe KPC tumor site, compared to a liposomal equivalent. This includes facilitated drug loading as a result of van der Waal’s forces, hydrogen bonding and electrostatic interactions with the wall of the silicasome pores. Improved drug delivery was accompanied by increased tumor cell killing at the primary and metastatic sites, as shown in the lower panel. Adapted from ref . Copyright 2016 American Chemical Society. Panel C: The silicasome carrier does not induce the bone marrow cytopenia, intestinal villi blunting and liver toxicity seen with free or a liposome encapsulated Irinotecan. Similar efficacy and safety have also been demonstrated in colon cancer models. Adapted from ref . Copyright 2016 American Chemical Society. Panel D: Silicasome uptake into the KPC tumor matrix and cancer cells was improved by co-administration of the cyclic iRGD peptide, which promotes transcytosis. Silicasomes were synthesized with an imageable gold nanoparticle core, followed by IV administration of the particles at 50 mg/kg in animals bearing orthotopic KPC tumors. Tumor tissues were collected for electron microscopy viewing after 24 hours. D-1 shows conventional and pseudocolor TEM images, demonstrating intravesicular particle transport across the tumor blood vessel wall. The vesicle numbers increased in animals receiving either separate injections or injection of the iRGD-conjugated nanocarrier. Adapted with permission from ref . Copyright 2017 American Society for Clinical Investigation. D-2 shows TEM images that demonstrate endothelial vesicles and particle localization in the tumor stroma or localized inside cancer cells. Adapted with permission from ref . Copyright 2017 American Society for Clinical Investigation. D-3 is a schematic to show the working mechanism of iRGD -induced transcytosis, which involves cyclic peptide binding to overexpressed integrins at the tumor site, peptide cleavage, and release of a CendR motif that activates the tyrosine protein kinase receptor, neurophilin-1. Adapted with permission from ref . Copyright 2018 Elsevier. This transcytosis mechanism is likely identical to the vesiculovascular organ, delineated by Nagy and Dvorek et al., who performed extensive EM analysis of multiple cancer types in humans (D-4).– Reprinted with permission from ref under the terms of the Creative Commons Attribution 2.0 License. Copyright 2008 The Authors.
Figure 8.
Figure 8.. Upscale production of Irinotecan-silicasomes for effective and safe treatment of PDAC.
Panel A: We have developed upscale production of an MSNP carrier, where the LB is used for Irinotecan (IRIN) remote loading, following encapsulation of sucralose octasulfate (TEA8SOS) in the particle pores.–, – Large batch production was made possible by using ethanol precipitation for LB coating instead of sonicating a biofilm, which has limitations for coating large particle batches. The upscale flow-through sonication procedure involves the direct introduction of an aqueous suspension of MSNP into a concentrated, ethanol suspended lipid solution, followed by controlled energy input in a flow cell sonication device. The coating mechanism is assembly of the suspended lipid monomers onto on the particle surfaces upon introduction into the aqueous environment. This approach is advantageous from the perspective that there is complete and rapid surface coating by the LB (cryoEM visualization) and avoidance of potentially toxic chloroform use. This approach allows the application of LB coating to 120 g MSNP batch sizes. The picture displays the average size dimensions and physicochemical characteristics, including IRIN loading capacity of ~40%. Adapted from ref . Copyright 2019 American Chemical Society. Panel B: Improved IRIN delivery and treatment efficacy in an orthotopic KPC model, using a silicasome vs. a liposome. The inserted table shows that the increased stability of the supported lipid bilayer improve carrier stability, circulatory half-life, leakiness and drug delivery atthe KPC tumor site, compared to a liposomal equivalent. This includes facilitated drug loading as a result of van der Waal’s forces, hydrogen bonding and electrostatic interactions with the wall of the silicasome pores. Improved drug delivery was accompanied by increased tumor cell killing at the primary and metastatic sites, as shown in the lower panel. Adapted from ref . Copyright 2016 American Chemical Society. Panel C: The silicasome carrier does not induce the bone marrow cytopenia, intestinal villi blunting and liver toxicity seen with free or a liposome encapsulated Irinotecan. Similar efficacy and safety have also been demonstrated in colon cancer models. Adapted from ref . Copyright 2016 American Chemical Society. Panel D: Silicasome uptake into the KPC tumor matrix and cancer cells was improved by co-administration of the cyclic iRGD peptide, which promotes transcytosis. Silicasomes were synthesized with an imageable gold nanoparticle core, followed by IV administration of the particles at 50 mg/kg in animals bearing orthotopic KPC tumors. Tumor tissues were collected for electron microscopy viewing after 24 hours. D-1 shows conventional and pseudocolor TEM images, demonstrating intravesicular particle transport across the tumor blood vessel wall. The vesicle numbers increased in animals receiving either separate injections or injection of the iRGD-conjugated nanocarrier. Adapted with permission from ref . Copyright 2017 American Society for Clinical Investigation. D-2 shows TEM images that demonstrate endothelial vesicles and particle localization in the tumor stroma or localized inside cancer cells. Adapted with permission from ref . Copyright 2017 American Society for Clinical Investigation. D-3 is a schematic to show the working mechanism of iRGD -induced transcytosis, which involves cyclic peptide binding to overexpressed integrins at the tumor site, peptide cleavage, and release of a CendR motif that activates the tyrosine protein kinase receptor, neurophilin-1. Adapted with permission from ref . Copyright 2018 Elsevier. This transcytosis mechanism is likely identical to the vesiculovascular organ, delineated by Nagy and Dvorek et al., who performed extensive EM analysis of multiple cancer types in humans (D-4).– Reprinted with permission from ref under the terms of the Creative Commons Attribution 2.0 License. Copyright 2008 The Authors.
Figure 9.
Figure 9.. Immunogenic Cell Death (ICD).
Panel A: ICD is a form of regulated cell death that activates immune responses, and of great interest in converting immune depleted or “cold” tumor microenvironments to immune inflamed or “hot”.–, ICD represents a functionally unique response pattern that comprises the induction of organellar and cellular stress, culminating in an apoptosis-like death response that is accompanied by the active secretion or passive release of numerous danger-associated molecular patterns (DAMPs)., , –, , –, The principal DAMPs are calreticulin (CRT), heat shock proteins, HMGB1, ATP, and cytokines (type I IFNs and IL-1 family). A number of chemotherapy agents are included in the list of ICD-inducing drugs, including anthracyclines (Doxorubicin, Idarubicin), Mitoxantrone, Bleomycin, Cyclophosphamide, Oxaliplatin, Paclitaxel and Irinotecan. Most of their pharmacologic actions include damage to the cell nucleus and DNA, with collateral effects on cellular stress pathways, including oxidative stress responses, endoplasmic reticulum (ER) stress, mitochondria, autophagy flux, and cell membrane affects, all contributing to DAMPs release. In addition, other small molecule agents such as Bortezomib, cardiac glycosides, Patupilone, Septacidin, Shikonin, Vorinostat, and Wogonin can trigger ICD. Panel B: in order to understand the biology of the immunogenic effect, CRT translocation to the tumor cell surface provides an “eat me” signal, which enhances the uptake of dying tumor cells by APCs, such as dendritic cells (DC). This leads to processing of endogenous tumor antigens, which are displayed to naïve T cells via Type I major histocompatibility complexes on the APC surface. Additional release of adjuvant stimuli, such as HMGB1 from the disintegrating cancer cell nuclei and ATP from autophagic vesicles play a role in DC recruitment and maturation. Panel C: It has been suggested that there are at least two different ICD response pathways: (i) chemotherapeutic agents and drugs that exert their primary effect on the nucleus, with collateral effects on cellular stress and DAMPs release (type I pathway); (ii) irradiation, hypericin-based photodynamic therapy and oncolytic viral stimuli, which primarily promotes cell stress responses, with secondary effects on apoptotic cell death (type II pathway).. Adapted with permission from ref . Copyright 2015 The International Journal of Developmental Biology. All considered, therapeutic use of the ICD pathway is to promote an endogenous tumor vaccination response, which may need to be boosted or propagated to account for the heterogeneity of the immune landscape.
Figure 10.
Figure 10.. Use of the Irinotecan-silicasome for PDAC chemo-immunotherapy by an ER stress pathway
Panel A: In addition to its action as a topoisomerase I inhibitor, Irinotecan (IRIN) induces a robust cell stress response because of its weak basic properties, which induces lysosomal alkalization and interference in autophagy flux. This induces oxidative stress and ER stress. The response is also accompanied by PD-L1 expression in KPC cells. Panel B: The IRIN-silicasome induces an ICD response in orthotopic KPC mice, injected IV on 3 occasions with either free IRIN or the IRIN-silicasome (40 mg/kg), followed by sacrifice 72 h later. IHC analysis, with the assistance of Aperio ImageScope software, was used to determine CRT and HMGB1 release (top) or recruitment of CD8+ and Foxp3+ cells (bottom) to the tumor landscape. Imaging intensity was quantitatively expressed as fold-increase compared to the saline group. Data are expressed as mean ± SEM, n = 3. *p < 0.05; **p < 0.01; ***p < 0.001 (1-way ANOVA followed by a Tukey’s test). Panel A-B reprinted with permission from ref under a Creative Commons Attribution License 4.0 (CC BY). Copyright 2021 The Authors.
Figure 10.
Figure 10.. Use of the Irinotecan-silicasome for PDAC chemo-immunotherapy by an ER stress pathway
Panel A: In addition to its action as a topoisomerase I inhibitor, Irinotecan (IRIN) induces a robust cell stress response because of its weak basic properties, which induces lysosomal alkalization and interference in autophagy flux. This induces oxidative stress and ER stress. The response is also accompanied by PD-L1 expression in KPC cells. Panel B: The IRIN-silicasome induces an ICD response in orthotopic KPC mice, injected IV on 3 occasions with either free IRIN or the IRIN-silicasome (40 mg/kg), followed by sacrifice 72 h later. IHC analysis, with the assistance of Aperio ImageScope software, was used to determine CRT and HMGB1 release (top) or recruitment of CD8+ and Foxp3+ cells (bottom) to the tumor landscape. Imaging intensity was quantitatively expressed as fold-increase compared to the saline group. Data are expressed as mean ± SEM, n = 3. *p < 0.05; **p < 0.01; ***p < 0.001 (1-way ANOVA followed by a Tukey’s test). Panel A-B reprinted with permission from ref under a Creative Commons Attribution License 4.0 (CC BY). Copyright 2021 The Authors.
Figure 11.
Figure 11.. Synthesis of a DACHPt-silicasome for PDAC chemo-immunotherapy.
Panel A: The DACHPt carrier is synthesized using the coordination chemistry of the drug under mild alkaline conditions. The panel on the left indicates that pH adjustment to obtain weak alkaline conditions increases the number of silanol groups in the particle pores. This allows electrostatic attachment of cationic DACHPt with achievement 22% loading capacity (compared to 4% for passively loaded Oxaliplatin). The bottom right panel demonstrates X-ray spectroscopy confirmation of the presence of Si, phosphorus (phospholipid) and Pt, also demonstrating that the Pt/Si ratio (w/w) is higher for DACHPt incorporation than Oxaliplatin. Panel B: The panel on the left shows a vaccination response of KPC cells exposed to 20 μm Doxorubicin, 500 μm Oxaliplatin, and 500 μm DACHPt. These agents induce an ICD response, which was confirmed by CRT expression after 24H (upper panel). Data is expressed as mean ± SD, n = 3. *p < 0.05 compared to PBS control. The left and lower panels show the vaccination response in which B6129SF1/J mice received subcutaneous administration of the chemo-treated KPC cells in one flank on two occasions, followed by injecting live KPC cells on the contralateral side. Tumor sizes were assessed at the challenge site on day 26, demonstrating an effective vaccination response to the chemo agents. It was also possible to show the generation of an ICD response in the orthotopic KPC model, with DACHPt being more effective than Oxaliplatin. The panel on the right demonstrates a KPC survival study, where animals treated with DACHPt silicasome at a Pt dose equivalent of 2 mg/kg IV every 3–4 days, in combination with IP administration of 100 μg anti-PD-1 antibody on 4 administrations. Saline, anti-PD-1 only, Oxaliplatin and Oxaliplatin plus anti-PD1 served as controls. Kaplan–Meier plots were used to display animal survival, which was significantly improved by DACHPt silicasome plus-anti-PD1 (n = 5–7, *p< 0.05. Log Rank test). Panel A-B adapted with permission from ref . Copyright 2021 John Wiley and Sons.
Figure 11.
Figure 11.. Synthesis of a DACHPt-silicasome for PDAC chemo-immunotherapy.
Panel A: The DACHPt carrier is synthesized using the coordination chemistry of the drug under mild alkaline conditions. The panel on the left indicates that pH adjustment to obtain weak alkaline conditions increases the number of silanol groups in the particle pores. This allows electrostatic attachment of cationic DACHPt with achievement 22% loading capacity (compared to 4% for passively loaded Oxaliplatin). The bottom right panel demonstrates X-ray spectroscopy confirmation of the presence of Si, phosphorus (phospholipid) and Pt, also demonstrating that the Pt/Si ratio (w/w) is higher for DACHPt incorporation than Oxaliplatin. Panel B: The panel on the left shows a vaccination response of KPC cells exposed to 20 μm Doxorubicin, 500 μm Oxaliplatin, and 500 μm DACHPt. These agents induce an ICD response, which was confirmed by CRT expression after 24H (upper panel). Data is expressed as mean ± SD, n = 3. *p < 0.05 compared to PBS control. The left and lower panels show the vaccination response in which B6129SF1/J mice received subcutaneous administration of the chemo-treated KPC cells in one flank on two occasions, followed by injecting live KPC cells on the contralateral side. Tumor sizes were assessed at the challenge site on day 26, demonstrating an effective vaccination response to the chemo agents. It was also possible to show the generation of an ICD response in the orthotopic KPC model, with DACHPt being more effective than Oxaliplatin. The panel on the right demonstrates a KPC survival study, where animals treated with DACHPt silicasome at a Pt dose equivalent of 2 mg/kg IV every 3–4 days, in combination with IP administration of 100 μg anti-PD-1 antibody on 4 administrations. Saline, anti-PD-1 only, Oxaliplatin and Oxaliplatin plus anti-PD1 served as controls. Kaplan–Meier plots were used to display animal survival, which was significantly improved by DACHPt silicasome plus-anti-PD1 (n = 5–7, *p< 0.05. Log Rank test). Panel A-B adapted with permission from ref . Copyright 2021 John Wiley and Sons.
Figure 12.
Figure 12.. Mitoxantrone-delivering liposomes induce an ICD response that can be augmented by co-delivery of an IDO-1 inhibitor.
Panel A: Two liposomes were constructed, one containing MTO-only (L-MTO), the second including a combination of MTO plus a prodrug IDO-1 inhibitor. While details about synthesis of the dual delivery liposome are discussed in Figure 18, this passage will provide a side-by-side comparison to maintain interpretation inclusive. The lipid composition of the L-MTO liposome was comprised of DSPC, Chol, CHEMS, and DSPE-PEG2kDa in the molar ratio of 45: 30: 20: 5, while the L-MTO/IND liposome contained DSPC: Chol-IND: CHMS: DSPE-PEG2kDa in the molar ratio of 45: 30: 20: 5. Liposome synthesis was carried out through hydration of a coated biofilm in a round bottom flask, followed by sonication in a citric acid buffer. Free soluble MTO was remotely loaded as described by us, followed by size exclusion chromatography to remove unencapsulated MTO. The purified L-MTO liposomes had an average diameter of ~112 nm with a low polydispersity index at 0.017 and a final drug loading at 9.7% (drug/lipid w/w). Comparable values for the L-MTO/IND liposome were the size of ~100 nm with a polydispersity index at 0.014 and a zeta-potential at −11.7 mV (detailed in Figure 18B). The presence of a drug precipitate in these liposomes is demonstrated in the cryoEM (upper panel). Panel B: L-MTO and L-MTO/IND were administered IV to mice with orthotopic 4T1 tumors to deliver an MTO equivalent dose of 3 mg/kg/injection, with/without the co-delivery of IND at 3 mg/kg/injection. The first injection was on day 8 when tumor size was 100–150 mm3, followed by 3 injections on days 11, 14, and 17. Mice were followed for 23 days. L-MTO administration induced significant tumor shrinkage, further enhanced by IND co-delivery (as explained later in Figure 18C). IHC analysis confirmed significant CRT and perforin expression (similar effects for HMGB1 and granzyme B are shown in Figure S10). Panel C: Different from the immunogenic effects of Doxorubicin and Irinotecan, the robust immunogenic effect of MTO does not include CTL recruitment, resulting instead in the generation of NKp46+ cells, which are particularly relevant for BC immunotherapy (Figure S16). Further data regarding the dual delivery liposome appear in Figure 18. Panel A-C adapted from ref . Copyright 2020 American Chemical Society.
Figure 12.
Figure 12.. Mitoxantrone-delivering liposomes induce an ICD response that can be augmented by co-delivery of an IDO-1 inhibitor.
Panel A: Two liposomes were constructed, one containing MTO-only (L-MTO), the second including a combination of MTO plus a prodrug IDO-1 inhibitor. While details about synthesis of the dual delivery liposome are discussed in Figure 18, this passage will provide a side-by-side comparison to maintain interpretation inclusive. The lipid composition of the L-MTO liposome was comprised of DSPC, Chol, CHEMS, and DSPE-PEG2kDa in the molar ratio of 45: 30: 20: 5, while the L-MTO/IND liposome contained DSPC: Chol-IND: CHMS: DSPE-PEG2kDa in the molar ratio of 45: 30: 20: 5. Liposome synthesis was carried out through hydration of a coated biofilm in a round bottom flask, followed by sonication in a citric acid buffer. Free soluble MTO was remotely loaded as described by us, followed by size exclusion chromatography to remove unencapsulated MTO. The purified L-MTO liposomes had an average diameter of ~112 nm with a low polydispersity index at 0.017 and a final drug loading at 9.7% (drug/lipid w/w). Comparable values for the L-MTO/IND liposome were the size of ~100 nm with a polydispersity index at 0.014 and a zeta-potential at −11.7 mV (detailed in Figure 18B). The presence of a drug precipitate in these liposomes is demonstrated in the cryoEM (upper panel). Panel B: L-MTO and L-MTO/IND were administered IV to mice with orthotopic 4T1 tumors to deliver an MTO equivalent dose of 3 mg/kg/injection, with/without the co-delivery of IND at 3 mg/kg/injection. The first injection was on day 8 when tumor size was 100–150 mm3, followed by 3 injections on days 11, 14, and 17. Mice were followed for 23 days. L-MTO administration induced significant tumor shrinkage, further enhanced by IND co-delivery (as explained later in Figure 18C). IHC analysis confirmed significant CRT and perforin expression (similar effects for HMGB1 and granzyme B are shown in Figure S10). Panel C: Different from the immunogenic effects of Doxorubicin and Irinotecan, the robust immunogenic effect of MTO does not include CTL recruitment, resulting instead in the generation of NKp46+ cells, which are particularly relevant for BC immunotherapy (Figure S16). Further data regarding the dual delivery liposome appear in Figure 18. Panel A-C adapted from ref . Copyright 2020 American Chemical Society.
Figure 13.
Figure 13.. Design of dual-drug nanocarriers to deliver ICD stimuli plus immune modulators interfering in immune escape pathways or delivering adjuvant stimuli.
The schematic illustrates liposome design, making use of remote loading of ICD-inducing chemo drugs, combined with inclusion of immune modulatory lipid moieties and prodrugs into the LB. Natural and synthetic lipid compounds that can be included in the LB are elucidated in Table 3. Prodrug design, with cleavable linkers and utility to interfere in a variety of immune escape pathways, are discussed in Figures 14–17 as well as in online Figures S17–S22. The formation of the lipid bilayer can be accomplished by different techniques, including hydration and sonication of a coated lipid biofilm, microfluidic mixing of ethanol/lipid and aqueous laminar flow channels in a reaction chamber (e.g., NanoAssemblr™). We have also described the use of flow-through sonication in Figure 8A.
Figure 14.
Figure 14.. General strategies to synthesize lipid-drug conjugates.
Cholesterol derivatives (detailed in Fig. 15), phospholipids, and fatty acids (detailed in Figure 16), collectively provide a wide selection of chemical building blocks for synthesizing designer prodrug conjugates, most preferably through the formation of ester, amide, ether, disulfide, imine, and carbamate conjugations. These linkers are acid/redox-sensitive or subject to enzymatic (e.g., esterase or protease) cleavage for drug release at target sites. The various drug lipidation options open new gateways to nano-enabled drug/gene delivery through lipid-bilayer drug anchoring/embedding, improved pharmacokinetics for tumor delivery, reduced toxicity due to systematic exposure, and additional drug combinations through remote drug loading.
Figure 15.
Figure 15.. Strategies for synthesizing cholesteryl-conjugated prodrugs.
Cholesterol-conjugated (cholesteryl) prodrugs provide a different type of anchor for LB incorporation. Compared to lipids, cholesterol increases lipid bilayer rigidity and eliminates bilayer phase transition in a concentration-dependent way, thereby increasing liposomal stability and slowing drug release (if desired). Useful cholesteryl building blocks such as cholesterol/cholesteryl chloride, cholesteryl mercaptan, cholesteryl chloroformate, and cholesteryl hemisuccinate (CHEMS) are commercially available for conjugation to hydrophilic and hydrophobic drugs by reactions detailed in Figure 14.
Figure 16.
Figure 16.. Strategies for synthesizing fatty acid-conjugated prodrugs.
Panel A: Fatty acids are versatile design elements for prodrug synthesis. For example, the lipid tails could be saturated or unsaturated, providing different self-assembly properties and membrane rigidity. On the other hand, drugs could be conjugated to the hydrophilic fatty acid head or at the end of the hydrophobic lipid tails, depending on the desired hydrophobicity of the drug/prodrug molecular after the conjugation. Useful building blocks for lipid-head drug conjugations include fatty acid chlorides, fatty acids, fatty aldehyde, and fatty alcohol. Panel B: Thiol/mercapto, amino, and hydroxyl-modified fatty acids are commercially available for synthesizing lipid-tail drug conjugates, yielding cleavable disulfide, imine, and ester bonds for hydrophobic drugs.
Figure 17.
Figure 17.. The role of the indoleamine 2,3-dioxygenase (IDO-1) metabolic pathway in immune interference, including in the counter-regulatory IFN-γ response pathway.
Panel A: Schematic to explain the role of IDO-1 in immune metabolic TME regulation by converting tryptophan to kynurenine. The kynurenine excess and tryptophan depletion interfere in the mTOR pathway and P−S6 kinase activity but enhance the activation of a kinase, “general control nonderepressible 2” (GNC2), as well as the transcriptional activity of the aryl hydrocarbon receptor (AhR). The overall impact is decreased CTL activity, T-cell anergy and increased Treg production. In addition, the activation of increased IL-6 production by AhR is responsible for enhanced IDO-1 production. Panel B: Mechanistic explanation of the counter-regulatory effect of ICD-induced IFN-γ release, which leads to upregulated IDO-1 and PD-L1 expression and Treg generation. This also explains cooperation between the metabolic and receptor-mediated checkpoint pathways towards immune escape in immune landscapes with an IFN-γ genomic footprint. Panel A-B adapted from ref . Copyright 2020 American Chemical Society.
Figure 18.
Figure 18.. Co-delivery of the IDO-inhibiting cholesteryl-Indoximod prodrug synergizes with mitoxantrone in augmenting chemo-immunotherapy in animal TNBC tumor models.
The dual-delivery L-MTO/IND data presented here supplements the data discussion in Figure 12 to show how the liposome was constructed, in addition to discussing the impact on another TNBC model. Panel A: A cholesteryl-ester-conjugated prodrug using the non-competitive IDO-1 inhibitor, 1-methyl-D tryptophan (a.k.a. Indoximod/IND) was used for prodrug development, involving 4 steps: (i) Boc-protection of IND, yielding Boc-IND; (ii) conjugation of Boc-IND to cholesterol by Steglich esterification; (iii) removal of the Boc group; (iv) desalting to yield CholIND-NH2. This streamlined approach is scalable and capable of providing highly purified gramscale quantities. Prodrug structure was confirmed by mass spectrometry and NMR. Panel B: The prodrug was incorporated into a liposomal carrier, making use of cholesteryl hemisuccinate (CHEMS) to neutralize the cationic charge of the ionized Chol-IND-NH3+ prodrug at physiological pH. This yielded L-MTO/IND liposomes with the physicochemical characteristics and cryoEM imaging features depicted in the lower panel. Both the L-MTO and L-MTO/IND liposomes showed significant improvement in PK and circulatory half-life, compared to free MTO (right panel). Panel C: L-MTO/IND showed significant improvement in tumor growth in both the 4T1 and EMT6 TNBC models, compared to L-MTO. Moreover, the ICD effect was mediated by NK cells instead of CTLs. Highly efficient liposomal drug delivery at both tumor sites was reflected by the blue coloration of the tumor tissue due to MTO. Panel A-C adapted from ref . Copyright 2020 American Chemical Society.
Figure 18.
Figure 18.. Co-delivery of the IDO-inhibiting cholesteryl-Indoximod prodrug synergizes with mitoxantrone in augmenting chemo-immunotherapy in animal TNBC tumor models.
The dual-delivery L-MTO/IND data presented here supplements the data discussion in Figure 12 to show how the liposome was constructed, in addition to discussing the impact on another TNBC model. Panel A: A cholesteryl-ester-conjugated prodrug using the non-competitive IDO-1 inhibitor, 1-methyl-D tryptophan (a.k.a. Indoximod/IND) was used for prodrug development, involving 4 steps: (i) Boc-protection of IND, yielding Boc-IND; (ii) conjugation of Boc-IND to cholesterol by Steglich esterification; (iii) removal of the Boc group; (iv) desalting to yield CholIND-NH2. This streamlined approach is scalable and capable of providing highly purified gramscale quantities. Prodrug structure was confirmed by mass spectrometry and NMR. Panel B: The prodrug was incorporated into a liposomal carrier, making use of cholesteryl hemisuccinate (CHEMS) to neutralize the cationic charge of the ionized Chol-IND-NH3+ prodrug at physiological pH. This yielded L-MTO/IND liposomes with the physicochemical characteristics and cryoEM imaging features depicted in the lower panel. Both the L-MTO and L-MTO/IND liposomes showed significant improvement in PK and circulatory half-life, compared to free MTO (right panel). Panel C: L-MTO/IND showed significant improvement in tumor growth in both the 4T1 and EMT6 TNBC models, compared to L-MTO. Moreover, the ICD effect was mediated by NK cells instead of CTLs. Highly efficient liposomal drug delivery at both tumor sites was reflected by the blue coloration of the tumor tissue due to MTO. Panel A-C adapted from ref . Copyright 2020 American Chemical Society.
Figure 18.
Figure 18.. Co-delivery of the IDO-inhibiting cholesteryl-Indoximod prodrug synergizes with mitoxantrone in augmenting chemo-immunotherapy in animal TNBC tumor models.
The dual-delivery L-MTO/IND data presented here supplements the data discussion in Figure 12 to show how the liposome was constructed, in addition to discussing the impact on another TNBC model. Panel A: A cholesteryl-ester-conjugated prodrug using the non-competitive IDO-1 inhibitor, 1-methyl-D tryptophan (a.k.a. Indoximod/IND) was used for prodrug development, involving 4 steps: (i) Boc-protection of IND, yielding Boc-IND; (ii) conjugation of Boc-IND to cholesterol by Steglich esterification; (iii) removal of the Boc group; (iv) desalting to yield CholIND-NH2. This streamlined approach is scalable and capable of providing highly purified gramscale quantities. Prodrug structure was confirmed by mass spectrometry and NMR. Panel B: The prodrug was incorporated into a liposomal carrier, making use of cholesteryl hemisuccinate (CHEMS) to neutralize the cationic charge of the ionized Chol-IND-NH3+ prodrug at physiological pH. This yielded L-MTO/IND liposomes with the physicochemical characteristics and cryoEM imaging features depicted in the lower panel. Both the L-MTO and L-MTO/IND liposomes showed significant improvement in PK and circulatory half-life, compared to free MTO (right panel). Panel C: L-MTO/IND showed significant improvement in tumor growth in both the 4T1 and EMT6 TNBC models, compared to L-MTO. Moreover, the ICD effect was mediated by NK cells instead of CTLs. Highly efficient liposomal drug delivery at both tumor sites was reflected by the blue coloration of the tumor tissue due to MTO. Panel A-C adapted from ref . Copyright 2020 American Chemical Society.
Figure 19.
Figure 19.. Design of a series of Linrodostat prodrugs to boost nano-enabled chemoimmunotherapy.
The competitive IDO-1 inhibitor, Linrodostat (BMS-986205), does not provide access to ester, ether or amine bonding for prodrug design. However, it is possible to accomplish bio-cleavable prodrugs by establishing an (acyloxy)alkyl carbamate site. Linrodostat contains an amide that can be reacted with chloromethyl chloroformate to generate a chloromethyl linker, which reacts with the silver salts carboxylic acid groups associated with fatty acids, CHEMS, and DGS derivatives. This yields AgCl precipitates and the formation of (acyloxy)alkyl carbamate-conjugated prodrugs.
Figure 20.
Figure 20.. Interference in PD-1 expression by small molecule GSK3 inhibitors.
Panel A: Schematic depicting the PD-1/PD-L1 signaling axis, which is responsible for suppressing CTL killing through interference in T-cell antigen receptor (TCR) signal transduction. PD-1 is expressed on “exhausted” T-cells, leading to the recruitment of SHP2 phosphatase, which interferes in recruitment of signaling components that bind to the tyrosine-based motifs in post-TCR complex. This prevents the release of cytolytic granules. Constitutionally active GSK3 is responsible for preventing the transcriptional activation of the T-bet promoter (Tbx21). Panel B: Introduction of a GSK3 inhibitor allows restoration of T-bet expression and interference in activation of the PD-1 promoter (Pdcd1) complex. The disappearance of PD-1 from the cell surface restores TCR signal transduction, allowing tumor cell killing by CTLs. In this sense, the transcriptional suppression of PD-1 provides the same outcome as anti-PD-1 blocking antibodies. Panel A-B reprinted with permission from ref . Copyright 2021 Elsevier.
Figure 21.
Figure 21.. Use of a reporter gene assay to illustrate BMS-8 interference in PD-1/PD-L1 interactions, as well as the approach of constructing a prodrug.
BMS-8 was chosen from among a short list of SMI (Figure S23), capable of disrupting PD-1/PD-L1 interactions, to develop a prodrug for LB incorporation. Panel A: BMS-8 exhibits a carboxy group that can be used for cholesterol conjugation, using Steglich esterification, as described in Figure S25. Panel B: BMS-8 exhibits a core/scaffold structure that induces PD-L1 dimerization, which prevents its ability to bind to PD-1. The blocking action can be demonstrated by the Promega PD-1 Cell Based Assay, which utilizes a Jurkat cell line, stably transfected with a nuclear factor of activated T-cell (NFAT) luciferase reporter plus a copy of cell surface expressed PD-1. TCR ligation in the absence of PD-1 engagement induces NFAT-luc activity. However, the TCR signal is blocked by antigen presenting (aAPC) CHO-K1 cells, stably transfected with PD-L1 (Figure 20). The addition of SMI inhibitors of the PD-1/PD-L1 axis restores TCR signal transduction, providing a quantitative readout to assess the avidity binding interference as by BMS inhibitors or antibodies, as shown in Figure S24.
Figure 22.
Figure 22.. Demonstration of the efficacy of free and encapsulated BMS-8 in a subcutaneous KPC model.
Mice received subcutaneous implementation with KPC tumor cells on days zero, followed by tracking tumor volumes for 23 days (upper panel). Animals received IV Irinotecan (IRIN) injection at a dose of 40 mg/Kg on days 10, 13 and 16, followed by the administration of BMS-8, BMS-8 prodrug (PD), anti-PD-1 antibody, or a dual-delivery IRIN/BMS-8PD silicasome on days 12, 15 and 18 (doses appear in the upper panel). The silicasome was synthesized by prodrug incorporation into the LB and remote loading (sucralose sulfate). Carrier physicochemical characterization is shown in the panel on the left. Animals were sacrificed on day 23 to collect tumor tissues for assessment of cytotoxic killing (Figure S28) and CTL recruitment (Figure S28). Tumor size plotting (lower panel) shows effective tumor shrinking in response to free IRIN, an outcome that was significantly enhanced by anti-PD1, BMS8, and BMS8-PD, as well as the dual-delivery (Irinotecan/BMS8-PD) silicasome. There was no improvement in tumor reduction of encapsulated over free BMS-8 in spite of a statistically significant increase (p = 0.047) in the BMS-8 tumor drug content by encapsulated (1.24 μg/g) vs. free drug (1.10 μg/g) delivery.
Figure 23.
Figure 23.. Effective delivery of a GSK3 inhibitor and tumor growth inhibition, using a remote loaded silicasome carrier
Figure S29 describes the use of medicinal chemistry criteria to accomplish the selection and remote loading of the GSK3 inhibitor, AZD1080, in a silicasome, with the characteristics shown in panel A. After growing subcutaneous MC38 tumors to ~300 mm3, mice were injected IV with 5 mg/kg encapsulated or free AZD1080. Animals were sacrificed after 24 and 48 hours, and blood, tumors, livers, spleens, kidneys, lungs, and hearts were harvested for HPLC quantification of tissue AZD1080 concentration (panel B), as described by Allen et al. Significance was assessed by 1-way ANOVA, * p < 0.05, **** p < 0.0001, n = 4. Panel C: Following the establishment of subcutaneous MC38 growth, treatment with free AZD1080, sAZD1080, anti-PD-1 antibody, SB415286, or saline commenced 10 days after inoculation (n = 9 animals/group). The silicasome was injected IV to deliver AZD1080 at a dose of 5 mg/kg. The controls were animals receiving IP doses of 5, 8 and 4 mg/kg, respectively, of free AZD1080, SB415286 (another GSK3i) and anti-PD-1. Treatment was repeated every 3 days for a total of 3 administrations. Mice were sacrificed 4 days after the final treatment. Panel D: Flow cytometry analysis to determine PD-1 staining intensity on tumor-infiltrating CD8+ T-cells, demonstrating a significant decrease in staining intensity in tumor tissue from animals treated with the AZD1080-silicasome vs. other treatments. The data confirmed interference in PD-1 expression by the encapsulated drug (** p < 0.01, NS = not significant). Comparable outcomes were achieved in subcutaneous pancreatic cancer (KPC), CD26 (colon cancer) and lung cancer (LLC) tumors, shown in Figure S30. Panel A-D adapted with permission from ref . Copyright 2021 Elsevier.
Figure 24.
Figure 24.. Prodrug design of the A2AR Inhibitor, ZM241385, four nanocarrier delivery.
The A2A receptor provides another avenue of immune escape in the solid tumor immune landscape and can be inhibited by the non-xanthine antagonist ZM241385. We identified the phenolic hydroxyl group on this compound for prodrug design. This enables synthesis of ester or ether drug-linkages to fatty alcohols or cholesterol, as well as the possibility to conjugate the drug to fatty acid, DGS or CHEMS carboxyl-terminal groups. Not only does bilayer incorporation of these drug conjugates offer improvement of the unfavorable drug PK, but also allows the development of co-formulated multi-drug carriers.
Figure 25.
Figure 25.. Nano-formulated CXCR4 inhibitors suppress tumor metastases and intervene in CTL exclusion in orthotopic TNBC and PDAC tumor models.
Panel A: We utilized the weak-basic properties of a selected series of CXCR4 inhibitors (AMD3100, AMD3465, and AMD11070) to assess drug loading capacities across liposomal and silicasome LB. This was accomplished using triethylammonium sucrose octasulfate (TEA8SOS) or ammonium sulfate to achieve loading capacities of 8~20% in liposomes. CryoEM images of the liposomes used for TEA8SOS loading and accompanying physicochemical characteristics are shown. AMD11070 was chosen to construct a silicasome, using the same trapping agent. For lipid coating, 40 mg/mL of the purified, bare MSNPs were used to soak in 80 mM TEA8SOS, before the addition of a 50% (w/v) lipids mixture (DSPC/Chol/DSPE-PEG2000, in the molar ratio 3:2:0.15. Panel B: Orthotopic 4T1 tumors were established as described in Figures 6 and 18. These animals develop a high rate of lung metastasis. Liposomal Doxorubicin (DOX-NP) induces significant 4T1 shrinkage (bottom left panel), with evidence of an immunogenic response as shown in Figure S4–S6. Free AMD11070 alone also leads to tumor shrinkage, which was significantly enhanced when combined with DOX-NP. In addition, combination therapy with DOX-NP plus liposomal AMD11070 provided additional tumor size reduction, in addition to accomplishing the highest density of CTL recruitment (Figure S34). IVIS imaging of explanted animal lungs demonstrated significant reduction in metastases under all conditions where AMD11070 was used. Panel C: Similar analysis was carried out in the orthotopic EMT6 model, which is characterized by extensive CD8+ exclusion from the tumor core, even under basal growth conditions (Figure 6). In this setting, neither free nor L-AMD11070 was able to interfere in tumor growth. However, combination of free or encapsulated AMD11070 with DOX-NP contributed to growth inhibition, which did not differ significantly between free and encapsulated drugs. However, spatial analysis of the tumor landscape, demonstrated that the increased recruitment of CD8+ T-cells during co-administration of DOX-NP, showed a significant shift in cell distribution to the tumor core. These changes were also accompanied by a reduction in Treg recruitment to the tumor landscape. Panel: D: AMD11070-silicasomes were used to perform a PK study in 10–12-week-old female B6/129SF1/J mice bearing KPC tumors (left panel). The animals received IV injection of free AMD11070 or AMD11070-silicasome at a drug dose of 5 mg/kg, followed by collection of blood samples at 5 min, 1, 4, 24, and 48 hrs. After separation of the plasma fraction, the drug was extracted in an acidic methanol solution (0.1 mol/L phosphoric acid/methanol, 1:4 v/v). Drug content in the tumor tissue was obtained from KPC tumor bearing animals 24hr after drug administration. The PK data were analyzed by PKSolver software, using a one-compartment model. We also investigated treatment impact on tumor weight and the CD8/Treg ratio in orthotopic KPC tumor bearing mice, as shown in the right panel. Animals received 3 IV administrations on days 8, 11 and 14, using the formulations shown in the legend, or were left untreated. Animals were sacrificed on day 17. Orthotopic tumors were collected, weighed, and prepared for sectioning to perform mIHC analysis, as described in Figure 6. While tumor growth inhibition by IRIN was not increased by co-delivery of the AMD11070-silicasome, this treatment resulted in a significant increase in the CD8/FoxP3 ratio in combination treatment of Irinotecan silicasome. * p< 0.05 compared to saline.
Figure 25.
Figure 25.. Nano-formulated CXCR4 inhibitors suppress tumor metastases and intervene in CTL exclusion in orthotopic TNBC and PDAC tumor models.
Panel A: We utilized the weak-basic properties of a selected series of CXCR4 inhibitors (AMD3100, AMD3465, and AMD11070) to assess drug loading capacities across liposomal and silicasome LB. This was accomplished using triethylammonium sucrose octasulfate (TEA8SOS) or ammonium sulfate to achieve loading capacities of 8~20% in liposomes. CryoEM images of the liposomes used for TEA8SOS loading and accompanying physicochemical characteristics are shown. AMD11070 was chosen to construct a silicasome, using the same trapping agent. For lipid coating, 40 mg/mL of the purified, bare MSNPs were used to soak in 80 mM TEA8SOS, before the addition of a 50% (w/v) lipids mixture (DSPC/Chol/DSPE-PEG2000, in the molar ratio 3:2:0.15. Panel B: Orthotopic 4T1 tumors were established as described in Figures 6 and 18. These animals develop a high rate of lung metastasis. Liposomal Doxorubicin (DOX-NP) induces significant 4T1 shrinkage (bottom left panel), with evidence of an immunogenic response as shown in Figure S4–S6. Free AMD11070 alone also leads to tumor shrinkage, which was significantly enhanced when combined with DOX-NP. In addition, combination therapy with DOX-NP plus liposomal AMD11070 provided additional tumor size reduction, in addition to accomplishing the highest density of CTL recruitment (Figure S34). IVIS imaging of explanted animal lungs demonstrated significant reduction in metastases under all conditions where AMD11070 was used. Panel C: Similar analysis was carried out in the orthotopic EMT6 model, which is characterized by extensive CD8+ exclusion from the tumor core, even under basal growth conditions (Figure 6). In this setting, neither free nor L-AMD11070 was able to interfere in tumor growth. However, combination of free or encapsulated AMD11070 with DOX-NP contributed to growth inhibition, which did not differ significantly between free and encapsulated drugs. However, spatial analysis of the tumor landscape, demonstrated that the increased recruitment of CD8+ T-cells during co-administration of DOX-NP, showed a significant shift in cell distribution to the tumor core. These changes were also accompanied by a reduction in Treg recruitment to the tumor landscape. Panel: D: AMD11070-silicasomes were used to perform a PK study in 10–12-week-old female B6/129SF1/J mice bearing KPC tumors (left panel). The animals received IV injection of free AMD11070 or AMD11070-silicasome at a drug dose of 5 mg/kg, followed by collection of blood samples at 5 min, 1, 4, 24, and 48 hrs. After separation of the plasma fraction, the drug was extracted in an acidic methanol solution (0.1 mol/L phosphoric acid/methanol, 1:4 v/v). Drug content in the tumor tissue was obtained from KPC tumor bearing animals 24hr after drug administration. The PK data were analyzed by PKSolver software, using a one-compartment model. We also investigated treatment impact on tumor weight and the CD8/Treg ratio in orthotopic KPC tumor bearing mice, as shown in the right panel. Animals received 3 IV administrations on days 8, 11 and 14, using the formulations shown in the legend, or were left untreated. Animals were sacrificed on day 17. Orthotopic tumors were collected, weighed, and prepared for sectioning to perform mIHC analysis, as described in Figure 6. While tumor growth inhibition by IRIN was not increased by co-delivery of the AMD11070-silicasome, this treatment resulted in a significant increase in the CD8/FoxP3 ratio in combination treatment of Irinotecan silicasome. * p< 0.05 compared to saline.
Figure 25.
Figure 25.. Nano-formulated CXCR4 inhibitors suppress tumor metastases and intervene in CTL exclusion in orthotopic TNBC and PDAC tumor models.
Panel A: We utilized the weak-basic properties of a selected series of CXCR4 inhibitors (AMD3100, AMD3465, and AMD11070) to assess drug loading capacities across liposomal and silicasome LB. This was accomplished using triethylammonium sucrose octasulfate (TEA8SOS) or ammonium sulfate to achieve loading capacities of 8~20% in liposomes. CryoEM images of the liposomes used for TEA8SOS loading and accompanying physicochemical characteristics are shown. AMD11070 was chosen to construct a silicasome, using the same trapping agent. For lipid coating, 40 mg/mL of the purified, bare MSNPs were used to soak in 80 mM TEA8SOS, before the addition of a 50% (w/v) lipids mixture (DSPC/Chol/DSPE-PEG2000, in the molar ratio 3:2:0.15. Panel B: Orthotopic 4T1 tumors were established as described in Figures 6 and 18. These animals develop a high rate of lung metastasis. Liposomal Doxorubicin (DOX-NP) induces significant 4T1 shrinkage (bottom left panel), with evidence of an immunogenic response as shown in Figure S4–S6. Free AMD11070 alone also leads to tumor shrinkage, which was significantly enhanced when combined with DOX-NP. In addition, combination therapy with DOX-NP plus liposomal AMD11070 provided additional tumor size reduction, in addition to accomplishing the highest density of CTL recruitment (Figure S34). IVIS imaging of explanted animal lungs demonstrated significant reduction in metastases under all conditions where AMD11070 was used. Panel C: Similar analysis was carried out in the orthotopic EMT6 model, which is characterized by extensive CD8+ exclusion from the tumor core, even under basal growth conditions (Figure 6). In this setting, neither free nor L-AMD11070 was able to interfere in tumor growth. However, combination of free or encapsulated AMD11070 with DOX-NP contributed to growth inhibition, which did not differ significantly between free and encapsulated drugs. However, spatial analysis of the tumor landscape, demonstrated that the increased recruitment of CD8+ T-cells during co-administration of DOX-NP, showed a significant shift in cell distribution to the tumor core. These changes were also accompanied by a reduction in Treg recruitment to the tumor landscape. Panel: D: AMD11070-silicasomes were used to perform a PK study in 10–12-week-old female B6/129SF1/J mice bearing KPC tumors (left panel). The animals received IV injection of free AMD11070 or AMD11070-silicasome at a drug dose of 5 mg/kg, followed by collection of blood samples at 5 min, 1, 4, 24, and 48 hrs. After separation of the plasma fraction, the drug was extracted in an acidic methanol solution (0.1 mol/L phosphoric acid/methanol, 1:4 v/v). Drug content in the tumor tissue was obtained from KPC tumor bearing animals 24hr after drug administration. The PK data were analyzed by PKSolver software, using a one-compartment model. We also investigated treatment impact on tumor weight and the CD8/Treg ratio in orthotopic KPC tumor bearing mice, as shown in the right panel. Animals received 3 IV administrations on days 8, 11 and 14, using the formulations shown in the legend, or were left untreated. Animals were sacrificed on day 17. Orthotopic tumors were collected, weighed, and prepared for sectioning to perform mIHC analysis, as described in Figure 6. While tumor growth inhibition by IRIN was not increased by co-delivery of the AMD11070-silicasome, this treatment resulted in a significant increase in the CD8/FoxP3 ratio in combination treatment of Irinotecan silicasome. * p< 0.05 compared to saline.
Figure 25.
Figure 25.. Nano-formulated CXCR4 inhibitors suppress tumor metastases and intervene in CTL exclusion in orthotopic TNBC and PDAC tumor models.
Panel A: We utilized the weak-basic properties of a selected series of CXCR4 inhibitors (AMD3100, AMD3465, and AMD11070) to assess drug loading capacities across liposomal and silicasome LB. This was accomplished using triethylammonium sucrose octasulfate (TEA8SOS) or ammonium sulfate to achieve loading capacities of 8~20% in liposomes. CryoEM images of the liposomes used for TEA8SOS loading and accompanying physicochemical characteristics are shown. AMD11070 was chosen to construct a silicasome, using the same trapping agent. For lipid coating, 40 mg/mL of the purified, bare MSNPs were used to soak in 80 mM TEA8SOS, before the addition of a 50% (w/v) lipids mixture (DSPC/Chol/DSPE-PEG2000, in the molar ratio 3:2:0.15. Panel B: Orthotopic 4T1 tumors were established as described in Figures 6 and 18. These animals develop a high rate of lung metastasis. Liposomal Doxorubicin (DOX-NP) induces significant 4T1 shrinkage (bottom left panel), with evidence of an immunogenic response as shown in Figure S4–S6. Free AMD11070 alone also leads to tumor shrinkage, which was significantly enhanced when combined with DOX-NP. In addition, combination therapy with DOX-NP plus liposomal AMD11070 provided additional tumor size reduction, in addition to accomplishing the highest density of CTL recruitment (Figure S34). IVIS imaging of explanted animal lungs demonstrated significant reduction in metastases under all conditions where AMD11070 was used. Panel C: Similar analysis was carried out in the orthotopic EMT6 model, which is characterized by extensive CD8+ exclusion from the tumor core, even under basal growth conditions (Figure 6). In this setting, neither free nor L-AMD11070 was able to interfere in tumor growth. However, combination of free or encapsulated AMD11070 with DOX-NP contributed to growth inhibition, which did not differ significantly between free and encapsulated drugs. However, spatial analysis of the tumor landscape, demonstrated that the increased recruitment of CD8+ T-cells during co-administration of DOX-NP, showed a significant shift in cell distribution to the tumor core. These changes were also accompanied by a reduction in Treg recruitment to the tumor landscape. Panel: D: AMD11070-silicasomes were used to perform a PK study in 10–12-week-old female B6/129SF1/J mice bearing KPC tumors (left panel). The animals received IV injection of free AMD11070 or AMD11070-silicasome at a drug dose of 5 mg/kg, followed by collection of blood samples at 5 min, 1, 4, 24, and 48 hrs. After separation of the plasma fraction, the drug was extracted in an acidic methanol solution (0.1 mol/L phosphoric acid/methanol, 1:4 v/v). Drug content in the tumor tissue was obtained from KPC tumor bearing animals 24hr after drug administration. The PK data were analyzed by PKSolver software, using a one-compartment model. We also investigated treatment impact on tumor weight and the CD8/Treg ratio in orthotopic KPC tumor bearing mice, as shown in the right panel. Animals received 3 IV administrations on days 8, 11 and 14, using the formulations shown in the legend, or were left untreated. Animals were sacrificed on day 17. Orthotopic tumors were collected, weighed, and prepared for sectioning to perform mIHC analysis, as described in Figure 6. While tumor growth inhibition by IRIN was not increased by co-delivery of the AMD11070-silicasome, this treatment resulted in a significant increase in the CD8/FoxP3 ratio in combination treatment of Irinotecan silicasome. * p< 0.05 compared to saline.
Figure 26.
Figure 26.. Use of a ratiometric designed silicasome carrier for improved chemotherapy in a human PDAC xenograft.
The dysplastic PDAC stroma contributes to chemo resistance, including inactivation of the first-line drug Gemcitabine (GEM) by the stromal enzyme, cytidine deaminase (CDA). Since it has been shown that Paclitaxel (PTX) can decrease GEM uptake through oxidative stress-mediated stromal depletion, we opted for a silicasome carrier that can deliver PTX plus GEM. This was accomplished by ratiometric design of a nanocarrier that incorporates GEM in the porous interior, with a sublethal amount of hydrophobic PTX incorporated in the LB. Ratiometric design, as described in Figure S38, yielded a dual-delivery carrier that incorporates 25 wt% GEM with 2.5 wt% PTX. The 10:1 ratiometric delivery was responsible for decreasing the stromal abundance, while increasing uptake of activated GEM 13-fold. We also demonstrated that the sublethal PTX dose could achieve the stromal response by generating oxidative stress instead of cell killing. To demonstrate the in vivo efficacy, mice carrying subcutaneous PANC-1 human xenografts received IV injection of PTX/GEM-loaded silicasomes. Drug co-delivery provided more effective tumor shrinkage than the GEM-only carrier, free GEM, or free GEM plus Abraxane. Comparable tumor shrinkage required coadministration of 12 times the amount of free Abraxane. Adapted from ref . Copyright 2015 American Chemical Society.
Figure 27.
Figure 27.. Two-wave of therapy to improve KPC chemo delivery by stromal-vascular engineering.
The dysplastic PDAC stroma plays a role in limiting vascular access to the tumor site through the recruitment of stromal pericytes to the abluminal endothelial cell surface. This recruitment is mediated by a TGF-β signaling pathway (panels A and B). Panel A demonstrates that the dense PDAC stroma blocks vascular access of IV-injected NIR-labeled liposomes, which becomes entrapped between the endothelial cells (CD31 green fluorescent marker) and the pericytes (NG2 blue fluorescent marker). The schematic below that illustrates these relationships. Pericytes adhere to endothelial cells as a consequence of TGF-β production in the stroma, which engages TGF-β type I receptor kinase activity. LY364947 is a SMI of this kinase, and can be delivered to the PDAC site by H-bonding to the surface MSNPs, decorated with a PEG/polyethylenimine polymer (Panel C). The drug is released in the TME by the acidic stromal pH, which interferes in drug binding to the decorated particle surface (Panel B). Panel D: In vivo experimentation using the LY364947-MSNP as a first wave carrier, injected IV, to increase uptake of the NIR-labeled, GEM-delivering liposomes injected 60 minutes later. The IVIS imaging in the upper panel demonstrates subcutaneous KPC growth of luciferase transfected tumor cells. The middle panel shows fluorescence imaging of the same tumors injected with the NIR-labeled liposomes, with and without prior LY364947-MSNP administration. The increased fluorescence intensity at the tumor site during two-way therapy is as a result of increased lysosomal release into the tumor site, as shown in the explanted tumors following animal sacrifice. Panel A-D adapted from ref . Copyright 2013 American Chemical Society.
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