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Back to Journal »International Journal of Nanomedicine» Volume 12

Author Zhang B, Song YM, Wang TQ, Yang SM, Zhang J, Liu YJ, Zhang N, Garg S 

Published on April 7, 2017, the 2017 volume: 12 pages 2871-2886

DOI https://doi.org/10.2147/IJN.S129091

Single anonymous peer review

Editor who approved for publication: Dr. Lei Yang

Bo Zhang,1 Yunmei Song,2 Tianqi Wang,1 Shaomei Yang,1 Jing Zhang,1 Yongjun Liu,1 Na Zhang,1 Sanjay Garg2 1Department of Pharmacy, School of Pharmacy, Shandong University, Jinan City, Shandong Province, China; 2 University of South Australia Pharmacy and Center for Pharmaceutical Innovation and Development (CPID), School of Medical Sciences, Adelaide, South Australia, Australia Abstract: Nanocomposite medicine is becoming a topic of concern in cancer treatment, although its clinical transformation is still extremely limited and challenging . One of the main obstacles is that it is difficult to effectively co-deliver immiscible hydrophilic/hydrophobic drugs to the tumor site. The purpose of this study is to develop a co-loaded lipid emulsion (LE) to co-deliver immiscible hydrophilic/hydrophobic drugs to improve cancer treatment, and to explore the co-delivery capability between co-loaded LE and mixture formulations. A variety of oxaliplatin/irinotecan drug-phospholipid complexes (DPC) are formulated. Using DPC technology to prepare co-loaded LE to effectively encapsulate two drugs. Co-loaded LE showed uniform particle size distribution, required stability and simultaneous release profile in both drugs. Compared with the simple solution mixture and the single-loaded LE mixture, the co-loaded LE showed excellent anti-tumor activity. In addition, the co-loaded nanocarriers can more effectively co-deliver the two drugs to the same cell, and show an optimized synergistic effect. These results indicate that co-loaded LE may be an ideal formula for enhancing cancer treatment with potential applications. The comparison between co-loaded LE and admixture formulations has important implications for drug design aimed at co-delivering multiple drugs. Keywords: cancer, combination therapy, co-administration, lipid emulsion, drug-phospholipid complex

Cancer is a major public health problem worldwide, and its morbidity and mortality are increasing. 1 Combination chemotherapy is widely used in clinics. 2, 3 Its purpose is to improve the therapeutic effect and reduce drug resistance through different modes of action or targeting a single cancer pathway. 4-6 Considering that traditional clinical administration is just a simple cocktail therapy, the uncoordinated pharmacokinetics and uncontrolled release characteristics of different drugs limit their application, leading to uncertainty in treatment. 7,8 The synergistic drug combination can normalize the pharmacokinetics and pharmacodynamics of the active agent, control the simultaneous release of the target site and provide excellent therapeutic effects. 5,9

Several different types of nanocarriers have been developed to co-deliver multiple drugs, such as liposomes, micelles, and mesoporous silica. 10,11 Due to the special core/shell structure, liposomes can simultaneously encapsulate hydrophilic and hydrophobic drugs. 12,13 The micelles can be encapsulated and combined to load different goods. 14,15 Due to the adjustable pore size and large pore volume of mesoporous silica, different drugs can be trapped in the pores. 16,17 However, the clinical transformation of nanocarrier-based combination therapies is still very important5,9 In addition to physical and chemical variability, safety issues, and the complexity of regulatory and manufacturing issues,18 the main obstacle lies in the difficulty of achieving multiple Efficient combined delivery, accurate carrier ratio and controlled simultaneous release of chemotherapeutic drugs, especially for immiscible hydrophilic/hydrophobic drugs. 19-21

Therefore, the development of nanocarriers that can provide immiscible hydrophilic/hydrophobic drug co-delivery with high-efficiency and mature technology of industrial production are very important. With this in mind, lipid emulsions (LEs) are selected as the ideal carrier to achieve the co-delivery capability of multiple drugs and improve the effect of cancer treatment. LEs, also known as fat emulsions or lipid microspheres, usually include emulsifiers, co-emulsifiers, stabilizers and isotonicity regulators to form a uniform oil-in-water formulation. 22-24 LEs can wrap the drugs in the inner oil phase to avoid drug leakage, precisely control the loading ratio of multiple drugs, and deliver the drugs to the tumor site together. Compared with other emerging nanocarriers, LEs have multiple advantages, including mature manufacturing and scalability technologies, required stability, and reliable safety of excipients. 25,26 In addition, LEs have been widely used for decades, and there are many clinical products such as diazepam, vitamins, propofol, prostaglandins, etomidate, and flurbiprofen. 27–29

In order to evaluate the co-delivery characteristics of LE, oxaliplatin (OXA) and irinotecan (IRI) were selected as model drugs in this study. These two chemotherapeutics are widely used clinically to treat colorectal cancer, and according to the National Comprehensive Cancer Network (NCCN) guidelines, their combination has been recommended as first-line treatment. 30 These drugs are immiscible and have significantly different water solubility. The co-delivery of hydrophilic and hydrophobic chemotherapeutics is expected to be more challenging.

Another purpose of this study is to evaluate the difference between mixed single LE (separate loading mode) and co-loaded LE (synchronous loading mode). It is expected that the single-loaded preparation will be more controllable during the production process, and it will be easier to control the drug ratio through simple mixing. Since the same nanocarrier has similar release and biodistribution characteristics, it is expected that the delivery capabilities of the two drugs will closely match. In contrast, many previous studies have described that co-loaded nanoparticles exhibit superior anti-tumor effects compared to simple mixtures. 12,31-35 Therefore, the differences in the therapeutic effect and co-delivery ability between simultaneous loading and the sub-assembly methods are also compared, which will increase the understanding of the co-delivery of multiple drugs.

The purpose of this study is to develop LE as a nanocarrier for the co-delivery of immiscible hydrophilic/hydrophobic chemotherapeutics. The drug-phospholipid complex (DPC) technology is used to prepare oxaliplatin and irinotecan co-loaded LEs (OXA/IRI-LEs), and characterize and evaluate them through in vitro and in vivo tests. The physical and chemical properties of the emulsion were characterized in terms of particle size, zeta potential, morphology and stability. An in vitro release study was performed to study the simultaneous release profile of the two drugs. In order to evaluate the therapeutic effect, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to test the in vitro cytotoxicity, and when carrying CT- 26 of the cells carried out in vivo anti-tumor activity in BALB/c mice. Near-infrared fluorophore (NIRF) imaging is used to simulate the biodistribution of drugs. In order to evaluate the co-delivery ability between the co-loaded emulsion and the physical mixture formulation to the same cells, the in vitro cell uptake of frozen sections and the in vivo confocal laser scanning microscope (CLSM) image were analyzed.

OXA and IRI were purchased from Boyuan Pharmaceutical Co., Ltd. (Jinan, China) and Knowshine Pharmachemicals Inc. (Shanghai, China), respectively. Egg phosphatidylcholine (EPC) and Pluronic F68 are from AVT Pharmaceutical Technology Co., Ltd. (Shanghai, China) and Sigma-Aldrich (St. Louis, Missouri, USA), respectively. MTT was purchased from Solarbio® Life Science (Beijing, China). Medium chain triglycerides (MCT) were provided by Luoxin Pharmaceutical Co., Ltd. (Linyi, China). All other reagents are commercially available analytical grade or higher.

Murine colon cancer cells (CT-26) and human colon cancer cells (HCT-116) were purchased from the Chinese Academy of Sciences (Shanghai, China). Both cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin at 37°C in an environment containing 5% CO2 nourish.

Female BALB/c mice (body weight: 18±2 g) were provided by the Medical Animal Test Center of Shandong University (Jinan, China). The animals were fed a standard diet and allowed to drink water at will. All experiments were carried out in accordance with the Animal Management Regulations of the Ministry of Health of the People's Republic of China (Document No. 55 in 2001). This study and experiment were formally approved by the Animal Ethics Committee of Shandong University (201002050).

Oxaliplatin-phospholipid complex (OPPC): Dissolve 20 mg OXA and 300 mg EPC in 20 mL methanol/dichloromethane (DCM) (v/v 9:1) and react at 40°C3 Hours to form a complex. The organic solvent was removed using a rotary evaporator to obtain OPPC.

Irinotecan-phospholipid complex (IPPC): Dissolve 50 mg IRI and 200 mg EPC in 10 mL DCM, and react at 40°C for 3 hours to form a complex. The organic solvent was removed using a rotary evaporator to obtain IPPC.

The preparation method of the co-loaded emulsion is shown in Figure 1. OPPC and IPPC were re-dissolved in DCM, 2g MCT and 50mg oleic acid (OA) were added; the DCM was removed using a rotary evaporator to obtain an oil phase. A total of 200 mg Pluronic F68 and 800 mg glycerin were dissolved in 40 mL of water to obtain an aqueous phase. The oil phase was added dropwise to the water phase under shear at 60°C. The formed coarse emulsion is further circulated through a high-pressure homogenizer (Panda 1K NS1001L; Niro Soavi SpA, Parma, Italy). For single-drug-loaded LE, only OPPC or IPPC was added and a similar preparation procedure was performed.

Figure 1 The preparation process of OXA/IRI co-loaded LE. Abbreviations: OXA, oxaliplatin; IRI, irinotecan; LE, lipid emulsion; OPPC, oxaliplatin-phospholipid complex; IPPC, irinotecan-phospholipid complex; DCM, dichloromethane; MCT, Medium chain triglycerides.

DPC is characterized by differential scanning calorimetry (DSC). The sample was sealed in an aluminum crucible and heated from 20°C to 350°C (TGA/SDTA851e; Mettler Toledo, Greifensee, Switzerland) at a flow rate of 50 mL/min in a nitrogen atmosphere. The DSC signal is recorded at a scan rate of 10°C per minute of heating.

The particle size distribution, polydispersity index (PDI) and Zeta potential of the emulsion were measured by dynamic light scattering using Zetasizer Nano ZS90 (Malvern, Worcestershire, UK). All measurements are performed in triplicate (n=3), and the values ​​are expressed as mean ± standard deviation (SD).

The morphology of the emulsion was observed by a transmission electron microscope (TEM) (JEM-1200EX, Japan). The sample was added to the surface of the copper grid and stained with phosphotungstic acid (1%, w/v). The acceleration voltage is 120 kV.

The physical stability of the emulsion is evaluated based on the stability constant (Ke) value. 36 samples (1 mL) were centrifuged at 3,000 rpm for 15 minutes in a centrifuge (Eppendorf AG 22331, Hamburg, Germany). Take a sample (50 μL) from the bottom and dilute with deionized water. Measure the absorbance of the sample (A) using an ultraviolet-visible spectrometer (Persee TU-1810, Beijing, China) at a wavenumber of 500 nm. In addition, the same procedure was used to measure the absorbance of LE without centrifugation (A0). Ke uses formula (1) to calculate:

In order to evaluate the storage stability of the preparation, the co-loaded LE was sterilized through a 220 nm filter, and 0.5 mL of the sample was extracted into a different tube and stored in the refrigerator (4°C) or room temperature (20°C±2 ℃) ). Take out each sample on a predetermined number of days, and measure the size distribution and PDI using Zetasizer Nano ZS90 (Malvern).

The in vitro release of OXA and IRI from LE was determined by dialysis. Due to the poor solubility of IRI and the sinking requirements of the release test, Tween-80 (0.5% w/v) was added to phosphate buffered saline (PBS) (pH 7.4) as the release medium. 37 In short, 1 mL of sample is added to a dialysis bag and incubated with 20 mL of release medium in a plastic tube at 37°C. Within a predetermined time interval, take out 2 mL of release medium from the outside of the dialysis bag and replace it with fresh medium. The cumulative amount of OXA and IRI in the release medium was measured by high-performance liquid chromatography (HPLC) and ultraviolet spectrophotometer, respectively. All measurements are made in triplicate.

The in vitro cytotoxicity of different preparations was tested in CT-26 and HCT-116 cells using the MTT assay. The cells were seeded in a 96-well plate at a density of 5,000 cells per well, and 150 μL of RPMI-1640 medium was added. After overnight incubation, the cells were treated with each preparation and incubated for 48 hours. Add MTT (20 μL, 5 mg/mL) to each well and incubate for another 4 hours. The cell plate was centrifuged at 3000 rpm for 10 minutes, and the medium was discarded; 150 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. Use a microplate reader (Model 680; Bio-Rad, Hercules, CA, USA) to measure the absorbance of the resulting DMSO solution at a wavelength of 570 nm. Use equation (2) to calculate relative cell viability (%):

Among them, Acontrol and Asample represent the absorbance of the negative control and the sample, respectively.

The eight groups are set as 1) Blank Lipid Emulsion (Blank-LEs); 2) Oxaliplatin Solution (OXA-Sol); 3) Irinotecan Solution (IRI-Sol); 4) Oxaliplatin Lipid Emulsion (OXA-LE); 5) Irinotecan lipid emulsion (IRI-LE); 6) Oxaliplatin plus irinotecan solution (OXA/IRI-Sol); 7) Oxaliplatin lipid emulsion and Irinotecan Rinotecan lipid emulsion mixture (OXA-LE/IRI-LE) and 8) OXA/IRI-LE.

A female BALB/c mouse xenograft model carrying CT-26 was used to study the anti-tumor efficacy in vivo. A 0.1 mL cell suspension containing 1×106 CT-26 cells was injected subcutaneously into the right axilla of the mouse. After cell inoculation, allow solid tumors to grow ≥100 mm3. After 1 week, the mice were divided into nine groups (six mice in each group): 1) 5% glucose solution (control); 2) blank LE; 3) OXA- Sol; 4) IRI-sol; 5) OXA-LE; 6) IRI-LE; 7) OXA/IRI-Sol; 8) OXA-LE/IRI-LE and 9) OXA/IRI-LE.

According to our previous research and publications, the doses of OXA and IRI were chosen to be 5 and 12.5 mg/kg. 38-41 Each group of mice was injected with the above preparation via tail vein once a week for 3 weeks. After the first administration, the diameter of the tumor was measured with a caliper, and the weight of the mouse was measured with an electronic balance every 2 days. Three weeks later, the mice were sacrificed, the tumors were excised and weighed. Use the following equation (3) to calculate the tumor volume:

Where L and W represent the length and width of the tumor, respectively.

In vivo and in vitro NIRF imaging

Real-time NIRF imaging is used to observe the biodistribution of drugs. DiR was selected as the NIRF dye because of its excellent skin penetration ability. The DiR-loaded LE was prepared by the same method as before, and the final concentration of DiR was 50 μg/mL (Supplementary material).

Female BALB/c mice carrying CT-26 were used for NIRF imaging. When the tumor volume reached> 300 mm3, mice were given 0.2 mL of free DiR and DiR-loaded LE via tail vein injection. After 1, 4, 8 and 24 hours, the mice were anesthetized with 10% chloral hydrate (intraperitoneal injection) and imaged. At the end of imaging, the mice were sacrificed, and the heart, liver, spleen, lung, kidney, and tumor were harvested for further ex vivo imaging. Real-time NIRF images were acquired using Xenogen IVIS Lumina system (Caliper Life Sciences, Hopkinton, MA, USA) and ICG filter (excitation: 745 nm, emission: 835 nm, exposure time: 3 s). Use Living Image 3.1 software (Caliper Life Sciences) to analyze the results.

Choose DiI (red fluorescence) and DiO (green fluorescence) as fluorescent dyes for labeling LE. Prepare DiI and/or DiO loaded LE using the same procedure (supplementary material) described previously.

CT-26 cells and HCT-116 cells were cultured overnight. For DiI and DiO, add labeled LE to the cells at a final concentration of 5 μg/mL. The two groups were studied as 1) a mixed group: a mixture of DiI-LEs and DiO-LEs; and 2) a shared group: DiI and DiO shared LE. After incubation for 0.5, 2 and 4 hours, the cells were washed 3 times with cold PBS, fixed with 4% paraformaldehyde for 20 minutes, and stained with Hoechst 33342 for 15 minutes. A confocal laser scanning microscope (LSM-780; Carl Zeiss, Germany) was used to observe the uptake of co-carrying LE cells.

To quantify the cell uptake efficiency, after incubation with DiI and DiO-labeled LE, the cells were digested and resuspended in 0.1 mL PBS, and flow cytometry (FACS Calibur; BD Biosciences, San Jose, CA, USA) was used.

In order to evaluate the co-delivery ability of LE into tumors in vivo, frozen section observations were performed in female BALB/c mice carrying CT-26. A 0.1 mL cell suspension containing 1×106 CT-26 cells was injected subcutaneously into the right axilla of the mouse. When the tumor volume reached 200-300 mm3, mice were injected with 1) DiI and DiO mixed solution, 2) DiI-LE and DiO-LE mixture, and 3) DiI/DiO co-loaded LE. The concentration of DiI and DiO are both 125 μg/mL. After the mice were sacrificed 12 hours after the intravenous (iv) injection, the tumors were collected and frozen sectioned with a thickness of 10 μm. The nuclei were stained with 2-(4-amidinophenyl)-6-indoleaminoformamidine dihydrochloride (DAPI), and then imaged using a confocal laser scanning microscope (LSM 780; Carl Zeiss).

All experimental data are expressed as mean ± SD. Student's t-test (Excel 2007, Microsoft) was used to evaluate statistical differences, and P<0.05 was considered statistically significant.

Both OPPC and IPPC were formulated for further drug loading, and the DSC thermogram was used to confirm the formation of the complex. DSC is a reliable method for screening the compatibility of drugs and excipients, and it provides the greatest information about possible interactions. 42,43 As shown in Figure 2, the DSC curves of OPPC and IPPC both show that the original peaks of the drug and phospholipids have disappeared. The significant difference between the pure drug and the complex indicates that the weak interaction between the drug and the phospholipid molecule, such as hydrogen bonds or van der Waals forces, leads to the formation of DPC. 44

Figure 2 DSC thermogram of DPC. Note: (A) OPPC; (B) International Plant Protection Convention. Abbreviations: DSC, differential scanning calorimetry; DPC, drug-phospholipid complex; OPPC, oxaliplatin-phospholipid complex; IPPC, irinotecan-phospholipid complex; OXA, oxaliplatin; EPC, egg Phospholipids; IRI, irinotecan.

Preparation and characterization of co-loaded LE

The optimized formula for co-loading LEs is listed in Table 1. Since MCT has better drug miscibility than long-chain oil, MCT is selected as the oil, and Pluronic F68 is added as an emulsifier in the water phase for better emulsification. Lipids and F68 as emulsifiers have been approved by the US Food and Drug Administration for intravenous injection (IV), indicating that the excipients have good safety. OA and glycerin are used as stabilizers and isotonic regulators, respectively. Based on our previous attempts, the mass ratio of OXA/IRI is optimized to 1:2.5, which is equal to the molar ratio of 1:1.5.38

Table 1 OXA/IRI co-loaded LEs optimized formula abbreviations: OXA, oxaliplatin; IRI, irinotecan; OA, oleic acid; LE, lipid emulsion; EPC, lecithin; MCT, medium chain triglycerides.

The particle size of all emulsion droplets is about 100 nm, and PDI <0.2 (Figure 3; Table 2), indicating that the LE prepared is uniform and has a narrow particle size distribution, which is suitable for intravenous injection. It is well known that the particle size of ~100-200 nm can promote vascular extravasation and interstitial transport to tumor problems through enhanced penetration and retention (EPR) effects. 45,46 The particle size of the co-loaded LE is slightly smaller than that of the single-loaded LE, which may be caused by the stereo specific occupancy. The morphology of the co-loaded LE in Figure 3C shows that the formulation is well dispersed and has a spherical shape. In addition, the average zeta potential of the prepared LE is about -20 mV; this negative charge will avoid recognition by the plasma and reduce the impact on blood clearance. 47

Figure 3 contains the characterization of LE, including particle size distribution (A), zeta potential (B) and TEM image (C). Note: The scale bar represents 200 nm. Abbreviations: LEs, lipid emulsion; TEM, transmission electron microscope.

Table 2 The particle size, zeta potential and stability constant of different lipid emulsions. Note: The data are expressed as mean ± SD. Abbreviations: PDI, polydispersity index; Ke, stability constant; OXA, oxaliplatin; LE, lipid emulsion; IRI, irinotecan.

The stability constant (Ke) is evaluated by the change in absorbance and is a quantitative method to determine the physical stability of the emulsion. The smaller the Ke value, the more stable the emulsion. Although the Ke value of co-loaded LE has increased slightly, it is still only about 10%, indicating that LE has good physical stability.

The storage stability of co-loaded LE was evaluated by measuring the particle size and the change of PDI for 91 days. The samples stored at 4°C are stable without significant changes in size and PDI. At the same time, the size of the sample under ambient temperature (20°C±2°C) significantly increased to nearly 300 nm after 91 days, indicating that the LE structure has changed (Figure 4). It was concluded that LE preparations should be stored at 4°C.

Figure 4 shows the changes in particle size (A) and PDI (B) of co-loaded LE after 91 days storage at 4°C and room temperature. Abbreviations: PDI, polydispersity index; LEs, lipid emulsion.

Dynamic membrane dialysis was used for in vitro release studies. As shown in Figure 5, OXA from OXA-LE and OXA/IRI-LE have similar release behavior: nearly 40% burst release within 30 minutes, followed by slow release, and approximately 80% release after 48 hours. Although IRI exhibits sustained release behavior at all time intervals without obvious initial burst release, due to its hydrophobicity, only 20% is released within 2 hours. The two drugs showed similar release profiles after 4 hours, indicating the simultaneous release of the two drugs.

Figure 5 In vitro release curves of OXA and IRI from single-carrying LE (A) and co-carrying LE (B). Note: Data is expressed as mean ± SD (n=3). Abbreviations: OXA, oxaliplatin; IRI, irinotecan; LE, lipid emulsion; SD, standard deviation.

We have previously confirmed that the greatest synergy exists in the IRI/OXA molar ratio of 1:1–1.5:1.38. The molar ratio of IRI/OXA released at different time intervals was further calculated to study the synergistic effect in this study (Table 3). The mixture and co-loaded LE can achieve the best synergy after 2 hours and 4 hours, respectively. The release rate of co-loaded LEs lags behind that of single LEs, which may be due to the competition between the two drugs to diffuse from the internal oil phase. On the other hand, the delayed release of co-loaded LE may help reduce leakage during cycling. Overall, these results show that compared with a single formulation, the co-loaded LE has the same release profile with almost no effect.

Table 3 Abbreviations of IRI/OXA molar ratio calculated at different time intervals: IRI, irinotecan; OXA, oxaliplatin; LEs, lipid emulsion.

The in vitro cytotoxicity of different preparations was evaluated by MTT assay (Figure 6). Blank LEs showed no obvious toxicity in CT-26 cells, while slight toxicity was observed in HCT-116 cells at higher concentrations. This difference may be due to different gene expression leading to different cell sensitivity, especially for different mouse-derived cells and human-derived cells. Compared with their solutions in two kinds of cells, OXA-LE and IRI-LE showed relative cytotoxicity. All the combination groups showed stronger cell killing ability than the single group. In addition, co-loaded LE, mixed solution and mixed LE showed similar cytotoxicity (P>0.05).

Figure 6 In vitro cytotoxicity study of different preparations on (A) CT-26 and (B) HCT-116 cells. Note: Data is expressed as mean ± SD (n=5). Abbreviations: SD, standard deviation; OXA, oxaliplatin; Blank-LEs, blank lipid emulsion; OXA-Sol, oxaliplatin solution; IRI-Sol, irinotecan solution; OXA-LEs, oxaliplatin Lipid emulsion; IRI-LEs, irinotecan lipid emulsion; OXA/IRI-Sol, oxaliplatin plus irinotecan solution.

According to the cell viability results (Table 4), the half maximum inhibitory concentration (IC50) value of the combination group calculated is lower than that of the single group, indicating the benefits of the combination strategy. Although the IC50 value of co-loaded LE was lower than that of mixture solution and mixture LE, no statistical difference was observed (P>0.05).

Table 4 Calculated IC50 values ​​of different preparations in CT-26 and HCT-116 cells Note: Data are expressed as mean ± SD (n=3). Abbreviations: IC50, half maximum inhibitory concentration; OXA-Sol, oxaliplatin solution; IRI-Sol, irinotecan solution; OXA/IRI-Sol, oxaliplatin plus irinotecan solution; OXA, oxaliplatin ; LE, lipid emulsion; IRI, irinotecan.

The in vivo anti-tumor efficacy of different formulations was evaluated in female BALB/c mice carrying CT-26. As shown in Figure 7, Blank-LEs did not show significant anti-tumor activity and had the same tumor volume compared with the control group. Although slight in vitro cytotoxicity was observed in HCT-116 cells, no tumor suppression or weight loss was observed in mice, which means that the nanocarriers are well tolerated.

Figure 7 In vivo anti-tumor activity of different preparations on female BALB/c mice carrying CT-26. Note: (A) tumor growth after intravenous administration in mice; (B) weight change of tumor-bearing mice; (C) photos of tumors separated after the mice were sacrificed; (D) histogram of tumor weight And (E) Histogram of net weight of mice. The frequency of dosing is marked with a black arrow. *P<0.05, **P<0.01, ***P<0.001, the difference is statistically significant compared with the control group; #P<0.05, ##P<0.01, ###P<0.001, which is comparable to co-loaded LE The ratio is statistically significant. Data are expressed as mean ± SD (n=6). Abbreviations: LEs, lipid emulsion; SD, standard deviation; OXA, oxaliplatin; Blank-LEs, blank lipid emulsion; OXA-Sol, oxaliplatin solution; IRI-Sol, irinotecan solution; OXA- LEs, oxaliplatin lipid emulsion; IRI-LEs, irinotecan lipid emulsion; OXA/IRI-Sol, oxaliplatin plus irinotecan solution; d, days.

For monotherapy, the anti-tumor activity of OXA-LEs and IRI-LEs is slightly better than that of corresponding drug solutions. OXA showed significant differences compared with the control group, and the P values ​​of the two IRI preparations were both >0.05. IRI did not show a significant therapeutic effect, because the selected mouse IRI dose was equivalent to that of the combined regimen and was not sufficient to use IRI alone to treat cancer. 38

In the case of combination therapy, the tumor growth of the combination group was inhibited compared with the single preparation. The co-loaded LEs (OXA/IRI-LEs) showed better anti-tumor activity than its solution (OXA/IRI-Sol) and mixture LEs (OXA-LEs plus IRI-LEs), with statistical significance (P<0.05) . The results also confirmed that compared with mixed LE and mixed solution, the weight of the tumor co-loaded with LE was much lower (P <0.05). On the other hand, compared with the control group, the co-loaded LE has a very significant difference, P<0.001, while the P value of the mixed solution and the mixed LE is <0.01.

The body weight of the mice was monitored to evaluate the safety of the preparation. As shown in Figure 7B, the body weight of the mice in each group showed an overall upward trend, and no serious weight loss was observed during the treatment. In addition, in order to eliminate the influence of tumor weight, the net weight of each group was calculated after adjusting the tumor weight. The weight of the co-loaded LE is higher than the mixed solution and mixed LE, indicating the safety of LE and minimal side effects (Figure 7E).

Biodistribution in the body is essential to explain the mechanism of enhanced anti-tumor effect. NIRF imaging is now widely accepted as one of the convenient methods for simulating the biodistribution of drugs in the body. 48,49 NIRF imaging is used to track the biodistribution of LE. As shown in Figure 8, most of the free DiR is distributed in the liver and spleen, but almost not in the tumor at all time intervals. For DiR-LEs, the liver is also the main accumulation organ, and the accumulation in tumors is significantly higher than that of free DiR. The NIRF signal of DiR-LEs persists in the tumor for 24 hours. The in vitro data directly illustrates the increase in the accumulation of the dye of the LE preparation in the tumor.

Figure 8 In vivo and in vitro NIRF imaging of tumor-bearing mice after intravenous injection of free DiR and DiR-labeled LE. Note: (A) In vivo imaging of mice at different time intervals, tumors are marked with red circles; (B) Ex vivo imaging of organs after dissection of mice 24 hours after administration. Abbreviations: DiR, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide; IV, intravenous injection; NIRF, near-infrared fluorophore; LEs, lipid emulsion.

Dyes are widely used in the pharmaceutical field as a substitute for tracking carriers to simulate in vitro and in vivo behaviors. 50-52 Assuming that the leakage of the dye from the carrier is inevitable, the in vitro release study of the two dyes was the first to study the loaded LE from a single or co-existence. As shown in Figure S1, the two dyes showed similar sustained-release curves from the prepared LE, and the leakage was <20% within 24 hours. The results showed that most of the dye remained in the inner oil phase of LEs, reflecting the distribution of the carrier.

The co-delivery efficiency of LE was evaluated by cell uptake behavior in CT-26 cells and HCT-116 cells. Cells incubated with LEs are positive for green (FL1-H, DiO) and red fluorescence (FL2-H, DiI). Due to the combination of green and red fluorescence, the co-delivery efficiency is represented by yellow fluorescence. As shown in Figure 9A and B, after 4 hours of incubation, both DiI and DiO cargo were distributed in the cytoplasm. The intensity of yellow fluorescence in each group increased with the increase of incubation time. The co-loaded LEs can see obvious strong yellow fluorescence, while compared with the co-loaded group, the mixed group can see more red and green fluorescence, indicating that the co-delivery efficiency of co-loaded LEs is higher. Similar results were obtained in CT-26 and HCT-116 cells.

Figure 9 In vitro cell uptake study of CT-26 cells (A and C) and HCT-116 cells (B and D) at different time intervals. Note: (A) and (B) fluorescence images. Red, green, and blue fluorescence represent DiI, DiO, and Hoechst 33342 staining, respectively, and yellow represents the combined image of red and green (magnification 63 times, bar represents 10 μm). (C) and (D) use flow cytometry for quantitative analysis. Abbreviation: LEs, lipid emulsion.

Flow cytometry was used to further quantify cellular uptake to assess the co-delivery capability of LE. In CT-26 cells (Figure 9C), the co-delivery efficiency of the co-loaded group was 25.2% after 0.5 h incubation, which was about 3.55 times higher than that of the mixed group (7.1%); at 2 h, the co-delivery efficiency of the mixed group was 68.4%. The group was 81.0%; and at 4 h, they were 84.7% and 93.0%, respectively, with no significant difference. Similar results also exist in HCT-116 cells. As shown in Figure 9D, the co-delivery efficiency (27.2%) of the co-loaded group was about 4.05 times higher than that of the mixed group (6.7%) at 0.5 hours, and even 1.76 times higher2 At h (37.2% in the mixture group, 65.5% in the total load group), 76.5% and 86.6% at 4 h, there was no significant difference.

To explore the ability of the preparation to co-deliver to tumors in vivo, CLSM images of tumor frozen sections were observed after administration of DiI and DiO-labeled preparations to mice. As shown in Figure 10, the fluorescence intensity of red and green in the solution group is much lower than that of the two LE groups. Obvious red and green can be seen in the solution group, indicating that the accumulation of free dye in the tumor is less than that of the LE preparation, and the distribution of free drug in the body is non-specific. Both LE groups showed more merged yellow than the solution, and the co-loaded LE had the top yellow in the merged picture, indicating that the two dyes accumulated better at the same site. These results are consistent with in vitro cell uptake studies, and show that co-carrying LE has an excellent ability to co-deliver two cargoes to the same tumor cell.

Figure 10: CLSM images of tumor frozen sections after administration of DiI and DiO mixed solution (lane 1), a mixture of DiI-LE and DiO-LE (lane 2) and DiI/DiO co-carrying LE (lane 3) to mice , Where red, green and blue represent the fluorescence of DiI, DiO and DAPI respectively (63 times magnification, and the bar represents 10 μm). Abbreviations: CLSM, confocal laser scanning microscope; DAPI, 2-(4-amidinophenyl)-6-indolocaramidine dihydrochloride; LE, lipid emulsion; DiI, red fluorescence; DiO, green fluorescence.

OXA/IRI co-loaded LE is prepared using DPC technology. DPC has been shown to be useful for enhancing lipophilicity and promoting the encapsulation of hydrophilic molecules into the hydrophobic core. Due to the molecular interaction between the drug and the phospholipid, it can also maintain drug release. 53 In this study, OXA, as a hydrophilic drug, is difficult to encapsulate in LE; therefore, it has proven to be very useful to convert it into a hydrophobic mode by producing OPPC. In our previous attempts, it was found that the IPPC in the formulation was more stable than the free IRI in the oil phase. In addition, multiple DPCs can coordinate the release profiles of different drugs, and achieve simultaneous release by reducing the difference in hydrophobicity between different drugs, thereby generating an optimized synergistic effect. Therefore, both DPCs were incorporated into the formulation for further study. Although many researchers have reported the use of DPC technology to control the sustained release of drugs, there are no reports about multiple DPCs to coordinate the simultaneous release of multiple drugs.

The co-loaded LE has the required size distribution and zeta potential and is suitable for IV injection. In vitro release studies have shown that both drugs can be simultaneously released from the co-loaded LE, which has the potential to optimize synergistic effects. In addition, compared with mixed solution and mixed LE, co-loaded LE exhibits excellent in vivo anti-tumor activity. All these results indicate that LEs will become excellent nanocarriers for co-delivery of multiple drugs. With mature technology, safe excipients and easy scale up, LE is a good choice to bridge the gap between unmet clinical needs and the uncertain transformational prospects of co-delivery of nanomedicine.

In vivo and in vitro NIRF imaging studies were carried out to illustrate the superior anti-tumor activity by simulating the biodistribution of the drug (Figure 8). The results showed that the drug accumulation of the LE preparation in the tumor was improved compared with the free drug. Although due to the difference between fluorescent dyes and drugs, this is only a preliminary assessment and cannot completely replace the real drug biodistribution, but it shows the accumulation capacity of tumors to a certain extent. These results indicate that LEs increase the accumulation of drugs in tumors, which will help enhance anti-tumor activity.

The cellular uptake process was further studied to explore the co-delivery efficiency of co-carrying LE (Figure 9). The early significant differences may be due to the competition between different nanoparticles in the endocytosis process. 54 Initially, different nanoparticles will be randomly distributed around the cell and then swallowed into the cytoplasm; therefore, the molar ratio of the two drugs in the same cell can be any ratio to produce individual therapeutic effects. At the later stage when the endocytosis process is close to saturation, the two LEs tend to be balanced and evenly distributed; therefore, the coexistence (yellow) in the cells can be observed. The difference in cell uptake behavior between the mixed group and the co-loaded group was finally reduced.

In vivo CLSM imaging of tumor frozen sections also proved that the co-loaded LE has a better co-delivery capability (Figure 10). Although the mixed group and the co-loaded group have the same chance of accumulating in the tumor, their endocytic processes are quite different. When a single load of LE mixture enters the cell, the molar ratio of the two drugs can be any ratio. At the same time, the co-loaded LEs will deliver the two drugs into the cells at a fixed ratio at the same time, so as to achieve an optimized synergistic effect. In short, we believe that the enhanced therapeutic effect of co-carrying LE is attributable to the higher efficiency of co-delivery of the two drugs into the same cell and the greatest synergy. All earlier results indicate that, due to excellent co-delivery capabilities, co-loaded formulas will be ideal carriers for the delivery of different goods.

OXA/IRI-LEs have been successfully developed to effectively encapsulate and coordinate the simultaneous release of two drugs. The in vitro release profile shows that both drugs can achieve sustained release from the co-loaded LE, and their molar ratio can be well controlled. Compared with solution and mixture LE, co-loaded LE exhibits excellent anti-tumor activity in vivo. In addition, co-carrying LE can more effectively co-deliver two drugs to the same cell, which may be essential to improve the therapeutic effect. These results confirm that OXA/IRI-LEs can be an effective agent for enhancing the treatment of colorectal cancer and have broad application prospects. The comparison between the co-loaded LE and the mixture group is valuable for drug design that co-delivers multiple drugs.

This work was supported by the China-Australia Health Science Research Center (CACHSR, 2014GJ10) and Shandong Science and Technology Development Project (2014GGE27121).

The authors report no conflicts of interest in this work.

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Preparation of Dye Loaded Lipid Emulsion (LE)

A total of 50 mg DiO (or DiI) was dissolved in 10 mL dichloromethane (DCM) and 250 mg egg phosphatidylcholine (EPC), and then reacted at 40°C for 3 hours to form a complex. Use a rotary evaporator to remove organic solvents to obtain DiO-DPC or DiI-DPC.

For the co-loaded LE, re-dissolve DiO-DPC and DiI-DPC in DCM, and add 2 g medium-chain triglycerides (MCT) and 50 mg oleic acid (OA); use a rotary evaporator to remove DCM to obtain oil Mutually. A total of 200 mg Pluronic F68 and 800 mg glycerin were dissolved in 40 mL of water to obtain an aqueous phase. After that, the oil phase was added dropwise to the water phase under shear at 60°C. The formed coarse emulsion is further circulated through a high-pressure homogenizer (Panda 1K NS1001L; Niro Soavi SpA, Parma, Italy).

For a single LE, re-dissolve DiO-DPC (or DiI-DPC) in DCM and add an additional 250 mg EPC, 2 g MCT, and 50 mg OA. Perform the same procedure in the following operations.

1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) loaded LE

A total of 50 mg DiR was dissolved in 10 mL DCM and 250 mg EPC, and then reacted at 40°C for 3 hours to form a complex. The organic solvent was removed using a rotary evaporator to obtain DiR-DPC.

Re-dissolve DiR-DPC in DCM and add an additional 250 mg EPC, 2 g MCT, and 50 mg OA. The DCM was removed using a rotary evaporator to obtain an oil phase. A total of 200 mg Pluronic F68 and 800 mg glycerin were dissolved in 40 mL of water to obtain an aqueous phase. After that, the oil phase was added dropwise to the water phase under shear at 60°C. The formed coarse emulsion is further circulated through a high-pressure homogenizer (Panda 1K NS1001L; Niro Soavi SpA).

Release DiI/DiO from LE in vitro

The in vitro release of DiI and DiO from LE was determined by dialysis. Tween-80 0.5% (w/v) was added to phosphate buffered saline (PBS) (pH 7.4) as the release medium required by the tank conditions for the release test. In short, add 1 mL of sample to a dialysis bag and incubate with 20 mL of release medium in a plastic tube at 37°C. Within a predetermined time interval, take out 2 mL of release medium and replace with fresh medium. Use a fluorescence spectrophotometer (F-7000, HITACHI, Japan) to measure the cumulative amount of DiI and DiO in the release medium. All measurements are performed twice.

Figure S1 The in vitro release curves of DiI and DiO in different LEs using PBS (pH 7.4) containing 0.5% Tween 80 (w/v) as the release medium. Note: (A) DiI is released from DiI-LEs, and DiO is released from DiO-LEs. (B) DiI and DiO are released from the co-loaded DiI/DiO-LE. Abbreviations: DiI, red fluorescence; DiO, green fluorescence; LE, lipid emulsion; PBS, phosphate buffered saline.

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