Improved Efficacy and Reduced Toxicity Using a Custom-Designed Irinotecan-Delivering Silicasome for Orthotopic Colon Cancer

Abstract

Irinotecan is a key chemotherapeutic agent for the treatment of colorectal (CRC) and pancreatic (PDAC) cancer. Because of a high incidence of bone marrow and gastrointestinal (GI) toxicity, Onivyde (a liposome) was introduced to provide encapsulated irinotecan (Ir) delivery in PDAC patients. While there is an ongoing clinical trial (NCT02551991) to investigate the use of Onivyde as a first-line option to replace irinotecan in FOLFIRINOX, the liposomal formulation is currently prescribed as a second-line treatment option (in combination with 5-fluorouracil and leucovorin) for patients with metastatic PDAC who failed gemcitabine therapy. However, the toxicity of Onivyde remains a concern that needs to be addressed for use in CRC as well. Our goal was to custom design a mesoporous silica nanoparticle (MSNP) carrier for encapsulated irinotecan delivery in a robust CRC model. This was achieved by developing an orthotopic tumor chunk model in immunocompetent mice. With a view to increase the production volume and to expand the disease applications, the carrier design was improved by using an ethanol exchange method for coating of a supported lipid bilayer (LB) that entraps a protonating agent. The encapsulated protonating agent was subsequently used for remote loading of irinotecan. The excellent irinotecan loading capacity and stability of the LB-coated MSNP carrier, also known as a "silicasome", previously showed improved efficacy and reduced toxicity when compared to an in-house liposomal carrier in a PDAC model. Intravenous injection of the silicasomes in a well-developed orthotopic colon cancer model in mice demonstrated improved pharmacokinetics and tumor drug content over free drug and Onivyde. Moreover, improved drug delivery was accompanied by substantially improved efficacy, increased survival, and reduced bone marrow and GI toxicity compared to the free drug and Onivyde. We also confirmed that the custom-designed irinotecan silicasomes outperform Onivyde in an orthotopic PDAC model. In summary, the Ir-silicasome appears to be promising as a treatment option for CRC in humans based on improved efficacy and the carrier's favorable safety profile.

Keywords: Onivyde; colorectal cancer; irinotecan; mesoporous silica nanoparticles; pancreatic cancer; silicasome; supported lipid bilayer.

Conflict of interest statement

Competing financial interests: The authors declare the following competing financial interest(s): Andre E. Nel and Huan Meng are co-founders and equity holders in Westwood Bioscience Inc. The remaining authors declare no conflict of interest.

Figures

Figure 1. Development of a custom-designed irinotecan silicasome nanocarrier.

(A) Schematic to show the different steps for developing the irinotecan nanocarrier, namely: (1) bare mesoporous silica nanoparticle (MSNP) synthesis and purification (see detailed description of the high-volume process online and Figures S2-8), (2) lipid coating of the particles containing the soaked-in trapping agent, triethylammmonium sucrose octasulfate (TEA8SOS); and (3) remote loading of irinotecan by a proton gradient (generated by the trapping agent), followed by purification and sterilization. (B) The final product, the Ir-silicasome, is comprised of a MSNP core that contains a large packaging space for irinotecan, which is stably entrapped by a lipid bilayer (LB). The LB contains a PEG attachment to improve colloidal stability and circulatory half-life. (C) Schematic to show the custom-designed procedure for surface coating by an alcohol-exchange method. Lipids are dissolved in ethanol as described in the online data section Figure S1. This ethanol suspension is rapidly mixed with TEA8SOS laden particles and sonicated, which leads to the lipids assembling on the particle surface, and rapid sealing of the pores. (D) The integrated synthesis process, with precise control of temperature, stirring speed, and addition of the precursor materials at optimal ratios, is capable of producing 18 L batches that contain ~100 g of particles, as described online. The table shows the physicochemical properties of the purified bare MSNPs. (E) CryoEM visualization of the Ir-silicasome and Onivyde®. The final Ir-silicasome product contains an irinotecan (free base) concentration of 4.3 mg/mL, which was dispensed in smaller volumes in glass containers. The table summarizes the comparative physicochemical properties.

Figure 1. Development of a custom-designed irinotecan silicasome nanocarrier.

(A) Schematic to show the different steps for developing the irinotecan nanocarrier, namely: (1) bare mesoporous silica nanoparticle (MSNP) synthesis and purification (see detailed description of the high-volume process online and Figures S2-8), (2) lipid coating of the particles containing the soaked-in trapping agent, triethylammmonium sucrose octasulfate (TEA8SOS); and (3) remote loading of irinotecan by a proton gradient (generated by the trapping agent), followed by purification and sterilization. (B) The final product, the Ir-silicasome, is comprised of a MSNP core that contains a large packaging space for irinotecan, which is stably entrapped by a lipid bilayer (LB). The LB contains a PEG attachment to improve colloidal stability and circulatory half-life. (C) Schematic to show the custom-designed procedure for surface coating by an alcohol-exchange method. Lipids are dissolved in ethanol as described in the online data section Figure S1. This ethanol suspension is rapidly mixed with TEA8SOS laden particles and sonicated, which leads to the lipids assembling on the particle surface, and rapid sealing of the pores. (D) The integrated synthesis process, with precise control of temperature, stirring speed, and addition of the precursor materials at optimal ratios, is capable of producing 18 L batches that contain ~100 g of particles, as described online. The table shows the physicochemical properties of the purified bare MSNPs. (E) CryoEM visualization of the Ir-silicasome and Onivyde®. The final Ir-silicasome product contains an irinotecan (free base) concentration of 4.3 mg/mL, which was dispensed in smaller volumes in glass containers. The table summarizes the comparative physicochemical properties.

Figure 1. Development of a custom-designed irinotecan silicasome nanocarrier.

(A) Schematic to show the different steps for developing the irinotecan nanocarrier, namely: (1) bare mesoporous silica nanoparticle (MSNP) synthesis and purification (see detailed description of the high-volume process online and Figures S2-8), (2) lipid coating of the particles containing the soaked-in trapping agent, triethylammmonium sucrose octasulfate (TEA8SOS); and (3) remote loading of irinotecan by a proton gradient (generated by the trapping agent), followed by purification and sterilization. (B) The final product, the Ir-silicasome, is comprised of a MSNP core that contains a large packaging space for irinotecan, which is stably entrapped by a lipid bilayer (LB). The LB contains a PEG attachment to improve colloidal stability and circulatory half-life. (C) Schematic to show the custom-designed procedure for surface coating by an alcohol-exchange method. Lipids are dissolved in ethanol as described in the online data section Figure S1. This ethanol suspension is rapidly mixed with TEA8SOS laden particles and sonicated, which leads to the lipids assembling on the particle surface, and rapid sealing of the pores. (D) The integrated synthesis process, with precise control of temperature, stirring speed, and addition of the precursor materials at optimal ratios, is capable of producing 18 L batches that contain ~100 g of particles, as described online. The table shows the physicochemical properties of the purified bare MSNPs. (E) CryoEM visualization of the Ir-silicasome and Onivyde®. The final Ir-silicasome product contains an irinotecan (free base) concentration of 4.3 mg/mL, which was dispensed in smaller volumes in glass containers. The table summarizes the comparative physicochemical properties.

Figure 2. Establishment of an orthotopic MC38-luc tumor chuck model in C57BL/6 mice.

(A) The orthotopic implantation involves minor surgery to place the MC38 tumor chunks on the cecum wall of C57BL/6 mice. Briefly, the tumor chunks were obtained from subcutaneous growing tumors established in C57BL/6 mice. Once the tumor reached ~1 cm in size, the tumor mass was aseptically harvested and cut up into 2~4 mm3 chunks. These tumor chunks were tied onto the cecum wall by absorbable surgical sutures. (B) H&E staining to show the growth of the orthotopic tumor in relation to the adjacent normal tissue. (C) Live-animal IVIS imaging to monitor the orthotopic tumor growth. The bioluminescence intensity was quantified at the region of interest (ROI) by IVIS Living Image software. (D) Example ex vivo IVIS image of the complete gastrointestinal tract of an animal, sacrificed ~3 weeks post tumor chunk implantation. More than 95% of operated mice developed primary tumors, which metastasized to adjacent intestinal tissues and the peritoneum.

Figure 2. Establishment of an orthotopic MC38-luc tumor chuck model in C57BL/6 mice.

(A) The orthotopic implantation involves minor surgery to place the MC38 tumor chunks on the cecum wall of C57BL/6 mice. Briefly, the tumor chunks were obtained from subcutaneous growing tumors established in C57BL/6 mice. Once the tumor reached ~1 cm in size, the tumor mass was aseptically harvested and cut up into 2~4 mm3 chunks. These tumor chunks were tied onto the cecum wall by absorbable surgical sutures. (B) H&E staining to show the growth of the orthotopic tumor in relation to the adjacent normal tissue. (C) Live-animal IVIS imaging to monitor the orthotopic tumor growth. The bioluminescence intensity was quantified at the region of interest (ROI) by IVIS Living Image software. (D) Example ex vivo IVIS image of the complete gastrointestinal tract of an animal, sacrificed ~3 weeks post tumor chunk implantation. More than 95% of operated mice developed primary tumors, which metastasized to adjacent intestinal tissues and the peritoneum.

Figure 2. Establishment of an orthotopic MC38-luc tumor chuck model in C57BL/6 mice.

(A) The orthotopic implantation involves minor surgery to place the MC38 tumor chunks on the cecum wall of C57BL/6 mice. Briefly, the tumor chunks were obtained from subcutaneous growing tumors established in C57BL/6 mice. Once the tumor reached ~1 cm in size, the tumor mass was aseptically harvested and cut up into 2~4 mm3 chunks. These tumor chunks were tied onto the cecum wall by absorbable surgical sutures. (B) H&E staining to show the growth of the orthotopic tumor in relation to the adjacent normal tissue. (C) Live-animal IVIS imaging to monitor the orthotopic tumor growth. The bioluminescence intensity was quantified at the region of interest (ROI) by IVIS Living Image software. (D) Example ex vivo IVIS image of the complete gastrointestinal tract of an animal, sacrificed ~3 weeks post tumor chunk implantation. More than 95% of operated mice developed primary tumors, which metastasized to adjacent intestinal tissues and the peritoneum.

Figure 3. Improved PK and tumor irinotecan concentrations using the silicasome carrier for treating orthotopic tumor-bearing mice.

(A) PK profile after a single IV injection of free drug or the nanocarriers at an irinotecan (IRIN) dose equivalent of 40 mg/kg (n = 3). Circulatory t1/2 values were calculated using PKSolver software. (B) Drug content at the tumor site after 48 hr and 72 hr in animals receiving an IV injection of 40 mg/kg irinotecan by the different carriers. (C) Ex vivo IVIS imaging of tumor-bearing mice receiving IV injection of DyLight680-labeled silicasomes at the identical dose in (A). Tumor tissue and major organs were harvested at 48 hr. (D) ICP-OES was used to quantify the percent injected Si dose (%ID) at the different sites after 48 hr. (E) Confocal microscopy to show the intratumoral distribution of the NIR silicasome particles used in the same experiment as in (C). Color code: Red, NIR silicasome particles; green, blood vessel staining with anti-CD31 antibody; blue, nuclear stained with DAPI. Bars represent 25 µm. Data represent mean ± SEM. *p<0.05 compared to saline; #p<0.05 compared to free IRIN; &p<0.05 compared to Onivyde® (1-way ANOVA followed by a Tukey’s test).

Figure 3. Improved PK and tumor irinotecan concentrations using the silicasome carrier for treating orthotopic tumor-bearing mice.

(A) PK profile after a single IV injection of free drug or the nanocarriers at an irinotecan (IRIN) dose equivalent of 40 mg/kg (n = 3). Circulatory t1/2 values were calculated using PKSolver software. (B) Drug content at the tumor site after 48 hr and 72 hr in animals receiving an IV injection of 40 mg/kg irinotecan by the different carriers. (C) Ex vivo IVIS imaging of tumor-bearing mice receiving IV injection of DyLight680-labeled silicasomes at the identical dose in (A). Tumor tissue and major organs were harvested at 48 hr. (D) ICP-OES was used to quantify the percent injected Si dose (%ID) at the different sites after 48 hr. (E) Confocal microscopy to show the intratumoral distribution of the NIR silicasome particles used in the same experiment as in (C). Color code: Red, NIR silicasome particles; green, blood vessel staining with anti-CD31 antibody; blue, nuclear stained with DAPI. Bars represent 25 µm. Data represent mean ± SEM. *p<0.05 compared to saline; #p<0.05 compared to free IRIN; &p<0.05 compared to Onivyde® (1-way ANOVA followed by a Tukey’s test).

Figure 3. Improved PK and tumor irinotecan concentrations using the silicasome carrier for treating orthotopic tumor-bearing mice.

(A) PK profile after a single IV injection of free drug or the nanocarriers at an irinotecan (IRIN) dose equivalent of 40 mg/kg (n = 3). Circulatory t1/2 values were calculated using PKSolver software. (B) Drug content at the tumor site after 48 hr and 72 hr in animals receiving an IV injection of 40 mg/kg irinotecan by the different carriers. (C) Ex vivo IVIS imaging of tumor-bearing mice receiving IV injection of DyLight680-labeled silicasomes at the identical dose in (A). Tumor tissue and major organs were harvested at 48 hr. (D) ICP-OES was used to quantify the percent injected Si dose (%ID) at the different sites after 48 hr. (E) Confocal microscopy to show the intratumoral distribution of the NIR silicasome particles used in the same experiment as in (C). Color code: Red, NIR silicasome particles; green, blood vessel staining with anti-CD31 antibody; blue, nuclear stained with DAPI. Bars represent 25 µm. Data represent mean ± SEM. *p<0.05 compared to saline; #p<0.05 compared to free IRIN; &p<0.05 compared to Onivyde® (1-way ANOVA followed by a Tukey’s test).

Figure 4. Comparative efficacy testing of the Ir-silicasome in the orthotopic MC38 model.

(A) A survival experiment was performed, in the course of which IVIS imaging was used to compare tumor growth up to day 21, beyond which metastatic peritoneal spread interfered in image detection. MC38 tumor-bearing mice (n = 6) received free irinotecan, Onivyde® or Ir-silicasome at an irinotecan dose equivalent of 40 mg/kg twice per week for up to six IV administrations. Saline was used as the negative control. Representative images are shown in the left panel, with quantitative data display of bioluminescence intensity at the ROI, using IVIS software. (B) Kaplan-Meier plots to display the survival rate of the different animal groups in the same experiment (*p<0.05, Log Rank test). (C) In a separate experiment, the tumor-bearing mice received similar doses as in (A) twice a week for a total of four administrations (n = 3). Animals were sacrificed at 24 hr after the last treatment (day 18). Orthotopic tumors were collected and weighed. (D) IHC analysis of cleaved caspase-3 (CC-3) expression in the orthotopic tumors harvested in (C). Quantification of the number of CC-3+ cells, using ImageScope software (right panel). Bar = 100 μm. Data represent mean ± SEM; *p<0.05 compared to saline; #p<0.05 compared to free IRIN; &p<0.05 compared to Onivyde®. “n.s.” indicates p>0.05.

Figure 4. Comparative efficacy testing of the Ir-silicasome in the orthotopic MC38 model.

(A) A survival experiment was performed, in the course of which IVIS imaging was used to compare tumor growth up to day 21, beyond which metastatic peritoneal spread interfered in image detection. MC38 tumor-bearing mice (n = 6) received free irinotecan, Onivyde® or Ir-silicasome at an irinotecan dose equivalent of 40 mg/kg twice per week for up to six IV administrations. Saline was used as the negative control. Representative images are shown in the left panel, with quantitative data display of bioluminescence intensity at the ROI, using IVIS software. (B) Kaplan-Meier plots to display the survival rate of the different animal groups in the same experiment (*p<0.05, Log Rank test). (C) In a separate experiment, the tumor-bearing mice received similar doses as in (A) twice a week for a total of four administrations (n = 3). Animals were sacrificed at 24 hr after the last treatment (day 18). Orthotopic tumors were collected and weighed. (D) IHC analysis of cleaved caspase-3 (CC-3) expression in the orthotopic tumors harvested in (C). Quantification of the number of CC-3+ cells, using ImageScope software (right panel). Bar = 100 μm. Data represent mean ± SEM; *p<0.05 compared to saline; #p<0.05 compared to free IRIN; &p<0.05 compared to Onivyde®. “n.s.” indicates p>0.05.

Figure 4. Comparative efficacy testing of the Ir-silicasome in the orthotopic MC38 model.

(A) A survival experiment was performed, in the course of which IVIS imaging was used to compare tumor growth up to day 21, beyond which metastatic peritoneal spread interfered in image detection. MC38 tumor-bearing mice (n = 6) received free irinotecan, Onivyde® or Ir-silicasome at an irinotecan dose equivalent of 40 mg/kg twice per week for up to six IV administrations. Saline was used as the negative control. Representative images are shown in the left panel, with quantitative data display of bioluminescence intensity at the ROI, using IVIS software. (B) Kaplan-Meier plots to display the survival rate of the different animal groups in the same experiment (*p<0.05, Log Rank test). (C) In a separate experiment, the tumor-bearing mice received similar doses as in (A) twice a week for a total of four administrations (n = 3). Animals were sacrificed at 24 hr after the last treatment (day 18). Orthotopic tumors were collected and weighed. (D) IHC analysis of cleaved caspase-3 (CC-3) expression in the orthotopic tumors harvested in (C). Quantification of the number of CC-3+ cells, using ImageScope software (right panel). Bar = 100 μm. Data represent mean ± SEM; *p<0.05 compared to saline; #p<0.05 compared to free IRIN; &p<0.05 compared to Onivyde®. “n.s.” indicates p>0.05.

Figure 4. Comparative efficacy testing of the Ir-silicasome in the orthotopic MC38 model.

(A) A survival experiment was performed, in the course of which IVIS imaging was used to compare tumor growth up to day 21, beyond which metastatic peritoneal spread interfered in image detection. MC38 tumor-bearing mice (n = 6) received free irinotecan, Onivyde® or Ir-silicasome at an irinotecan dose equivalent of 40 mg/kg twice per week for up to six IV administrations. Saline was used as the negative control. Representative images are shown in the left panel, with quantitative data display of bioluminescence intensity at the ROI, using IVIS software. (B) Kaplan-Meier plots to display the survival rate of the different animal groups in the same experiment (*p<0.05, Log Rank test). (C) In a separate experiment, the tumor-bearing mice received similar doses as in (A) twice a week for a total of four administrations (n = 3). Animals were sacrificed at 24 hr after the last treatment (day 18). Orthotopic tumors were collected and weighed. (D) IHC analysis of cleaved caspase-3 (CC-3) expression in the orthotopic tumors harvested in (C). Quantification of the number of CC-3+ cells, using ImageScope software (right panel). Bar = 100 μm. Data represent mean ± SEM; *p<0.05 compared to saline; #p<0.05 compared to free IRIN; &p<0.05 compared to Onivyde®. “n.s.” indicates p>0.05.

Figure 5. Reduction of bone marrow and GI tract toxicity by encapsulated irinotecan delivery by the silicasome.

(A) Peripheral blood was collected to obtain differential WBC and neutrophil counts in non-tumor-bearing animals 24 hr after receiving 4 IV injections of the various irinotecan formulations at 40 mg/kg. Bone marrow toxicity was evaluated by H&E staining of sternal tissue. Normalized total bone marrow cellularity was determined by using Aperio ImageScope software to calculate the surface area occupied by all cell types (middle panel), as well as the surface area occupied by nucleated hematopoietic cells (right panel). (B) Representative H&E images of the sternums. Both low (bar = 400 μm) and high (bar = 50 μm) magnification pictures are shown. (C) GI tract toxicity evaluated by IHC analysis to discern the number of intestinal groups displaying cleaved caspase-3 (CC-3). The intestines were collected from the experiment in (A). Representative CC-3 IHC staining images in low (bar = 100 μm) and high (bar = 50 μm) magnification are shown. (D) Quantitative display of the percentage CC-3+ cells. Data represent mean ± SEM. *p<0.05 compared to saline; #p<0.05 compared to free IRIN; &p<0.05 compared to Onivyde®.

Figure 5. Reduction of bone marrow and GI tract toxicity by encapsulated irinotecan delivery by the silicasome.

(A) Peripheral blood was collected to obtain differential WBC and neutrophil counts in non-tumor-bearing animals 24 hr after receiving 4 IV injections of the various irinotecan formulations at 40 mg/kg. Bone marrow toxicity was evaluated by H&E staining of sternal tissue. Normalized total bone marrow cellularity was determined by using Aperio ImageScope software to calculate the surface area occupied by all cell types (middle panel), as well as the surface area occupied by nucleated hematopoietic cells (right panel). (B) Representative H&E images of the sternums. Both low (bar = 400 μm) and high (bar = 50 μm) magnification pictures are shown. (C) GI tract toxicity evaluated by IHC analysis to discern the number of intestinal groups displaying cleaved caspase-3 (CC-3). The intestines were collected from the experiment in (A). Representative CC-3 IHC staining images in low (bar = 100 μm) and high (bar = 50 μm) magnification are shown. (D) Quantitative display of the percentage CC-3+ cells. Data represent mean ± SEM. *p<0.05 compared to saline; #p<0.05 compared to free IRIN; &p<0.05 compared to Onivyde®.

Figure 5. Reduction of bone marrow and GI tract toxicity by encapsulated irinotecan delivery by the silicasome.

(A) Peripheral blood was collected to obtain differential WBC and neutrophil counts in non-tumor-bearing animals 24 hr after receiving 4 IV injections of the various irinotecan formulations at 40 mg/kg. Bone marrow toxicity was evaluated by H&E staining of sternal tissue. Normalized total bone marrow cellularity was determined by using Aperio ImageScope software to calculate the surface area occupied by all cell types (middle panel), as well as the surface area occupied by nucleated hematopoietic cells (right panel). (B) Representative H&E images of the sternums. Both low (bar = 400 μm) and high (bar = 50 μm) magnification pictures are shown. (C) GI tract toxicity evaluated by IHC analysis to discern the number of intestinal groups displaying cleaved caspase-3 (CC-3). The intestines were collected from the experiment in (A). Representative CC-3 IHC staining images in low (bar = 100 μm) and high (bar = 50 μm) magnification are shown. (D) Quantitative display of the percentage CC-3+ cells. Data represent mean ± SEM. *p<0.05 compared to saline; #p<0.05 compared to free IRIN; &p<0.05 compared to Onivyde®.

Figure 5. Reduction of bone marrow and GI tract toxicity by encapsulated irinotecan delivery by the silicasome.

(A) Peripheral blood was collected to obtain differential WBC and neutrophil counts in non-tumor-bearing animals 24 hr after receiving 4 IV injections of the various irinotecan formulations at 40 mg/kg. Bone marrow toxicity was evaluated by H&E staining of sternal tissue. Normalized total bone marrow cellularity was determined by using Aperio ImageScope software to calculate the surface area occupied by all cell types (middle panel), as well as the surface area occupied by nucleated hematopoietic cells (right panel). (B) Representative H&E images of the sternums. Both low (bar = 400 μm) and high (bar = 50 μm) magnification pictures are shown. (C) GI tract toxicity evaluated by IHC analysis to discern the number of intestinal groups displaying cleaved caspase-3 (CC-3). The intestines were collected from the experiment in (A). Representative CC-3 IHC staining images in low (bar = 100 μm) and high (bar = 50 μm) magnification are shown. (D) Quantitative display of the percentage CC-3+ cells. Data represent mean ± SEM. *p<0.05 compared to saline; #p<0.05 compared to free IRIN; &p<0.05 compared to Onivyde®.

Figure 6. The custom designed Ir-silicasome demonstrate increased efficacy over Onivyde® in an orthotopic PDAC model.

(A) Intratumoral irinotecan content in orthotopic KPC tumor bearing mice that received a single IV injection of the Ir-silicasome, Onivyde®, or free drug at an irinotecan dose equivalent of 40 mg/kg. The mice were sacrificed after 48 hr or 72 hr, and irinotecan content at the harvested tumor sites was determined by UPLC-MS as described in Figure 3B. (B) Efficacy experiment to compare the effects of various irinotecan formulations on primary tumor growth and metastasis. Orthotopic KPC tumor bearing animals received treatments at an irinotecan dose of 40 mg/kg twice per week or saline, for a total of three IV administrations (n = 3). Animals were sacrificed at 24 h after the last treatment; autopsy and ex vivo bioluminescence imaging were performed to evaluate the primary and metastatic tumor burden in each group. IHC analysis of CC-3 was performed on primary tumors. (C) An independent experiment was conducted to determine the survival outcome between Ir-silicasome vs. Onivyde® (n = 8). Orthotopic KPC-bearing mice received IV injections of an equivalent dose of 40 mg/kg irinotecan twice per week for a total of six administrations. Overall survival rate was determined as described in Figure 4 (left bottom panel, *p<0.05, Log Rank test), and orthotopic tumor growth was monitored by live animal tumor bioluminescence imaging (Right bottom panel, Figure S18). Data represent mean ± SEM. *p<0.05 compared to saline; #p<0.05 compared to free IRIN; &p<0.05 compared to Onivyde®.

Figure 6. The custom designed Ir-silicasome demonstrate increased efficacy over Onivyde® in an orthotopic PDAC model.

(A) Intratumoral irinotecan content in orthotopic KPC tumor bearing mice that received a single IV injection of the Ir-silicasome, Onivyde®, or free drug at an irinotecan dose equivalent of 40 mg/kg. The mice were sacrificed after 48 hr or 72 hr, and irinotecan content at the harvested tumor sites was determined by UPLC-MS as described in Figure 3B. (B) Efficacy experiment to compare the effects of various irinotecan formulations on primary tumor growth and metastasis. Orthotopic KPC tumor bearing animals received treatments at an irinotecan dose of 40 mg/kg twice per week or saline, for a total of three IV administrations (n = 3). Animals were sacrificed at 24 h after the last treatment; autopsy and ex vivo bioluminescence imaging were performed to evaluate the primary and metastatic tumor burden in each group. IHC analysis of CC-3 was performed on primary tumors. (C) An independent experiment was conducted to determine the survival outcome between Ir-silicasome vs. Onivyde® (n = 8). Orthotopic KPC-bearing mice received IV injections of an equivalent dose of 40 mg/kg irinotecan twice per week for a total of six administrations. Overall survival rate was determined as described in Figure 4 (left bottom panel, *p<0.05, Log Rank test), and orthotopic tumor growth was monitored by live animal tumor bioluminescence imaging (Right bottom panel, Figure S18). Data represent mean ± SEM. *p<0.05 compared to saline; #p<0.05 compared to free IRIN; &p<0.05 compared to Onivyde®.

Figure 6. The custom designed Ir-silicasome demonstrate increased efficacy over Onivyde® in an orthotopic PDAC model.

(A) Intratumoral irinotecan content in orthotopic KPC tumor bearing mice that received a single IV injection of the Ir-silicasome, Onivyde®, or free drug at an irinotecan dose equivalent of 40 mg/kg. The mice were sacrificed after 48 hr or 72 hr, and irinotecan content at the harvested tumor sites was determined by UPLC-MS as described in Figure 3B. (B) Efficacy experiment to compare the effects of various irinotecan formulations on primary tumor growth and metastasis. Orthotopic KPC tumor bearing animals received treatments at an irinotecan dose of 40 mg/kg twice per week or saline, for a total of three IV administrations (n = 3). Animals were sacrificed at 24 h after the last treatment; autopsy and ex vivo bioluminescence imaging were performed to evaluate the primary and metastatic tumor burden in each group. IHC analysis of CC-3 was performed on primary tumors. (C) An independent experiment was conducted to determine the survival outcome between Ir-silicasome vs. Onivyde® (n = 8). Orthotopic KPC-bearing mice received IV injections of an equivalent dose of 40 mg/kg irinotecan twice per week for a total of six administrations. Overall survival rate was determined as described in Figure 4 (left bottom panel, *p<0.05, Log Rank test), and orthotopic tumor growth was monitored by live animal tumor bioluminescence imaging (Right bottom panel, Figure S18). Data represent mean ± SEM. *p<0.05 compared to saline; #p<0.05 compared to free IRIN; &p<0.05 compared to Onivyde®.