- Open Access
Specific drug delivery efficiently induced human breast tumor regression using a lipoplex by non-covalent association with anti-tumor antibodies
- Yu-Ling Lin†1View ORCID ID profile,
- Nu-Man Tsai2, 3,
- Chia-Hung Chen†4,
- Yen-Ku Liu5,
- Chung-Jen Lee6,
- Yi-Lin Chan7,
- Yu-Shan Wang8,
- Yuan-Ching Chang9,
- Chi-Hsin Lin10,
- Tse-Hung Huang11, 12, 13,
- Chao Ching Wang11,
- Kwan-Hwa Chi8Email author and
- Kuang-Wen Liao4, 5, 14, 15Email author
© The Author(s) 2019
- Received: 27 August 2018
- Accepted: 14 January 2019
- Published: 6 February 2019
A cationic liposome-PEG-PEI complex (LPPC) was employed as a carrier for achieving targeted delivery of drug to human epidermal growth factor receptor-2 (HER2/neu)-expressing breast cancer cells. LPPC can be easily loaded with an anti-tumor drug and non-covalently associated with an anti-tumor antibody such as Herceptin that is clinically used to rapidly form immunoparticles within 1 h.
Drug-loaded LPPC have an average size about 250 nm and a zeta potential of about 40 mV. Herceptin was complexed onto surface of the LPPC to form the drug/LPPC/Herceptin complexes. The size of curcumin/LPPC/Herceptin complexes were 280 nm and the zeta potentials were about 23 mV. Targeting ability of this delivery system was demonstrated through specific binding on surface of cells and IVIS images in vivo, which showed specific binding in HER2-positive SKBR3 cells as compared to HER2-negative Hs578T cells. Only the drug/LPPC/Herceptin complexes displayed dramatically increased the cytotoxic activity in cancer cells. Both in vitro and in vivo results indicated that Herceptin adsorbed on LPPC directed the immunocomplex towards HER2/neu-positive cells but not HER2/neu-negative cells. The complexes with either component (curcumin or doxorubicin) used in the LPPC-delivery system provided a better therapeutic efficacy compared to the drug treatment alone and other treatment groups, including clinical dosages of Herceptin and LipoDox, in a xenografted model.
LPPC displays important clinical implications by easily introducing a specific targeting characteristic to drugs utilized for breast cancer therapy.
- Lipo-PEG-PEI complex
- Drug delivery
Most doctors consider chemotherapy as an efficient and essential strategy of clinical therapy for patients with metastatic tumors. Anti-tumor drugs are used to inhibit tumor growth, suppress angiogenesis in tumors or induce apoptosis in tumor cells spread throughout the patient’s body. There is no doubt that the relative amount of anti-tumor drugs accumulated within the tumor area is crucial for therapeutic efficacy. Although the administration of high doses of anti-tumor drugs may indeed increase the levels of cytotoxicity in cancer cells, this also increases the risk of normal organ failure. Circumventing this issue, drug-encapsulated liposomes and other nanoparticles have been found to selectively target tumors via leaky vessels, thereby enhancing the anti-tumor efficacy and reducing the side effects in normal tissues [1–4]. Furthermore, covalent conjugation of the targeting molecules, such as with cell-specific peptides (RGD and GE11 peptides) [5–7] and monoclonal antibodies (anti-HER2/neu and anti-EGFR antibodies) [1, 8–10], has further improved the efficacy compared with drug-encapsulated particles.
The available targetable nanoparticles are produced using thiol, carboxylic acid or amine groups for the covalent linkage, which may attenuate the activity of certain targeting molecules during the coupling reaction [11–13]. Additionally, prior processing of the nanoparticle and targeting molecules requires multiple steps and are therefore both time-consuming and sample-consuming [8, 14, 15]. In contrast to covalent conjugation, the non-covalent method of targeting molecules with cationic nanoparticles may circumvent this issue by electrostatic interaction if the targeting molecules stably exist on nanoparticles. However, the weak interaction between the targeting molecules and nanoparticles is usually interrupted by certain substances within the microenvironment, including salts and proteins, which cause the defluxion of targeting molecules from the liposomes and influences the specific targeting [12, 16]. Previous studies have shown that IgG sonicated with phospholipids in which 4–40% of the IgG is bound to the vesicles, but the vesicle-bound IgG only retains low activities [17, 18]. To improve the system, cationic P(MDS-co-CES) micelles were developed, which bind Herceptin or TRAIL via hydrogen bonds and/or hydrophobic interactions and encapsulates doxorubicin or paclitaxel to target cancer cells in vitro [19–21]. Until now, the in vivo efficacy of this approach has not yet been displayed.
Chemicals and antibodies
Curcumin (≧ 94% purity), polyethylene glycol (PEG, MW 1500 and 8000), polyethyleneimine (branched PEI, MW 25,000) and 3,3′-dioctadecyloxacarbocyanine (DiO) were purchased from Sigma-Aldrich (St. Louis, MO). 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC) and 1,2-Dilauroyl-sn-Glycero-3-Phosphocholine (DLPC) were purchased from Avanti Polar Lipids (Alabaster, AL). Herceptin was purchased from Roche (Basel, Switzerland). Rabbit polyclonal antibody to human IgG F(c)-FITC was purchased from Acris Antibodies GmbH (Herford, Germany).
Preparation of the Lipo-PEI-PEG complex (LPPC)
LPPC was prepared as previously described. Briefly, 25 mg of DOPC and DLPC were mixed in 1 ml chloroform and coated onto a round bottom flask by a rotary evaporator (EYELA, N-1000S, Tokyo, Japan) at 37 °C to yield a thin lipid film. The lipid films were hydrated by steam for 2 h and then 5 ml of PEG-PEI solution (675 mg PEI and 220 mg PEG8000 in deionized water) were added into the container. The lipid films were vigorously resuspended for 10 min and then the suspension was extruded through a LiposoFast extruder (Avestin Inc., Ottawa, Canada) via a 200 nm mesh 9 times. The suspensions were diluted in deionized water 50-fold and centrifuged at 5900×g for 5 min to remove any unincorporated substances. Finally, the pellets were resuspended with deionized water and both types of particles, curcumin/LPPC and empty LPPC, were stored at 4 °C until needed. Before use, both types of lipoplex were warmed to room temperature.
The formation and characterization of the drug/LPPC/Herceptin complex
For drug encapsulation, 10 μl of 100 mM curcumin or 40 mg/ml Dox were mixed with 1 mg of LPPC at room temperature for 30 min. After incubation, the mixture of curcumin or Dox and LPPC were centrifuged at 5900×g for 5 min to remove the non-encapsulated drug. The curcumin concentration remaining in the supernatant of the solution was then measured using a spectrophotometer (Amersham Biosciences, Uppsala, Sweden) at 432 nm. The Dox concentration remaining in the supernatant of the solution was then measured using a fluorescent spectrophotometer (Hitachi, Tokyo, Japan) at Ex 470 nm/Em 590 nm. The pellets (curcumin/LPPC) were resuspended with 100 μl deionized water and stored at 4 °C.
For the adsorption of the targeting molecule, 40 μg of drug/LPPC was incubated with 200 μg of Herceptin (Roche, Basel, Switzerland) in 50 μl for 30 min. After incubation, the excess positive charges of the drug/LPPC/Herceptin complexes were reduced by PEG1500incubation for 30 min twice and centrifuged at 5900×g for 5 min to remove the excess PEG1500. The particle sizes and zeta potentials of the empty LPPC and curcumin/LPPC incorporated with Herceptin were determined using a Zetasizer instrument (Zetasizer 3000HS, Malvern Instruments, Malvern, UK). The measurements of 2 mg of the various LPPC complexes were taken in 200 μl deionized water at room temperature. The in vitro release of curcumin from the Curcumin/LPPC or Curcumin/LPPC/Herceptin complexes were determined as previously described .
Targeting ability of LPPC/Herceptin complexes in vitro
The HER2-positive cells, MDA-MB-231, MCF7 and SKBR3, and the HER2-negative Hs578T cell lines were obtained from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) and maintained according to the manufacturer’s instructions. These cell lines (3 × 105 cells) were incubated with Herceptin for 30 min followed by incubation for 30 min with fluorescein-conjugated rabbit anti-human IgG (Acris Antibodies GmbH, Herford, Germany; 1: 10,000). The fluorescence was assayed via flow cytometry (Becton–Dickinson, San Jose, CA).
LPPC was first labeled with 3 mM fluorescent lipophilic dye DiO (Sigma-Aldrich, St. Louis, MO; 10 μl in 1 mg LPPC at a final volume of 110 μl) for 30 min and subsequently washed and resuspended as described above. Next, DiO-incorporated LPPC (20 μg) was complexed with either 2 μg of Herceptin or 2 μg of Rituximab (anti-human CD20 antibody) and then blocked with 20 μl of PEG1500 (100 mg/ml) for an additional 30 min. Various human breast tumor cells (3 × 105 cells) were incubated with 20 μg of DiO/LPPC/Herceptin or DiO/LPPC/Rituximab at 4 °C for 30 min in the dark. After the cells were washed and resuspended in 1 ml DMEM, the cells were analyzed by a flow cytometry.
Intracellular accumulation of curcumin
MCF7 cells were seeded onto glass coverslips (Nunc, USA) at a density of 2 × 105 cells per disc overnight. The cells were treated with 2 ml of medium containing either curcumin, curcumin/LPPC/Rituximab or curcumin/LPPC/Herceptin at a final curcumin concentration of 2 μM. After incubation at 37 °C for 0.5, 1 or 2 h, the media was removed and the cells were washed with PBS, fixed with 4 w/w % paraformaldehyde in PBS, and imaged with a 400× magnification using a Zeiss LSM 510 META confocal microscope (Carl Zeiss, Thornwood, NY, USA).
Specific targeting ability of LPPC/Herceptin complexes in vivo
Athymic nude mice were inoculated with 2 × 107 Hs578T cells or SKBR3 cells via injection into the left and right flank of the back. Before treatment, 1 mg of LPPC was incorporated with 3 mM fluorescent lipophilic dye DiI as described above. DiI-incorporated LPPCs (100 μg) were complexed with 10 μg of antibodies for 30 min, blocked with PEG1500 for 30 min, and intravenously injected into the nude mice bearing tumors. Whole body imaging of the tumor-bearing mice was performed at various time points with an IVIS Spectrum System (Caliper Life Sciences Inc., Hopkinton, MA, USA). Finally, the mice were sacrificed, and the organs were collected and analyzed with an IVIS Spectrum System.
Cytotoxicity of curcumin/LPPC
The different cell lines were seeded onto 96-well tissue culture plates at a concentration of 1 × 104 cells/100 μl/well overnight. Subsequently, the cells were treated with serial concentrations of the various agents, empty LPPC, LPPC/Herceptin, curcumin/LPPC, curcumin/LPPC/Herceptin and non-encapsulated curcumin. On the other set of studies, the cells were treated with serial concentrations of the agents, like as Dox/LPPC, Dox/LPPC/Herceptin and non-encapsulated Dox. After 48 h of incubation, the levels of cell viability for each cell line was determined by MTT colorimetric assay. The cell viability values were plotted as a percentage of the untreated control.
Female BALB/c, BALB/c/nu and NOD-SCID mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and maintained on a 12:12-h light: dark cycle in an animal environmental control chamber (Micro-VENT IVC Systems, Allentown, NJ). Humane animal care was ensured by use of the institutional guidelines of National Chiao Tung University (NCTU). All animal studies were approved by the Institutional Animal Care and Use Committee in National Chiao Tung University (NCTU-IACUC-104034).
Antitumor activity of curcumin/LPPC in vivo
NOD-SCID mice were inoculated with 1 × 107 SKBR-3 cells via injection into the right flank. Once a tumor mass was established, the mice were intravenously injected with either PBS, Cur/LPPC/Rituximab (40 mg/kg curcumin and 4 mg/kg Rituximab), Cur/LPPC/Herceptin (40 mg/kg curcumin and 4 mg/kg Herceptin), high dose of Cur/LPPC/Herceptin (Cur-H/LPPC/Herceptin, 200 mg/kg curcumin and 4 mg/kg Herceptin) or Herceptin (9 mg/kg Herceptin, a clinical dose) once every 3 days. The tumor volumes were measured using a caliper every 2 days, and the tumor volume was calculated using the following formula: volume (mm3) = length × width × high. The survival were also measured every 2 days.
The tumor-bearing mice were treated with either PBS, Dox/LPPC/Herceptin (5 mg/kg Dox and 4 mg/kg Herceptin), high dose of Dox/LPPC/Herceptin (Dox-H/LPPC/Herceptin, 50 mg/kg Dox and 4 mg/kg Herceptin), Dox plus Herceptin (5 mg/kg Dox and 4 mg/kg Rituximab) or LipoDox plus Herceptin once every week as described above. Both the tumor growth and survival were assessed as described above.
The results were analyzed using the SAS statistical software package (SAS Institute Inc., Cary, USA). The results were expressed as the mean ± SD. A t test was used when comparing two independent samples, and the ANOVA test was used when comparing multiple samples. Differences in which p < 0.05 were considered statistically significant.
Characteristics of drug loaded LPPC/Herceptin
Anti-tumor drugs such as curcumin are easily loaded into LPPC by incubation at room temperature and remove the non-encapsulated curcumin by centrifugation. The maximal drug encapsulated capacity of 1 mg LPPC was approximately 321.6 μg curcumin (loading efficiency is 87.2%). The proteins carry capacity of Curcumin/LPPC reach 40 μg Herceptin as well as LPPC (Fig. 1b). The LPPC bound Herceptin was stable on the LPPC or Curcumin/LPPC (Additional file 1: Figure S1). Compared to the empty LPPC, the particle sizes of Cur/LPPC/Herceptin were slightly increased (Fig. 1c and Additional file 1: Table S1), but the charges of Cur/LPPC/Herceptin were dramatically decreased (Fig. 1d, and Additional file 1: Table S1). Figure 1e and Additional file 1: Figure S2 showed the encapsulated drug slowly released from LPPC and the adsorption of Herceptin did not affect drug release from LPPC.
Targeting ability of LPPC-based complexes in vitro and in vivo
To examine the specific targeting ability of the LPPC/Herceptin complex in vivo, DiI-labeled LPPC/Herceptin or LPPC/Rituximab complexes were used to probe HER2-positive tumors. The results in Fig. 2c show that only Herceptin but not Rituximab directed the DiI-labeled LPPC complexes to the SKBR3 tumors (HER2-positive) in 72 h, but both failed to result in significant fluorescence in Hs578T tumors (HER2-negative). Later, their organs or tumors were collected to further analysis. The results showed that Rituximab-based LPPC complexes caused the non-specific accumulations in organs including liver, stomach, intestine, and both tumors. In contrast to Rituximab, Herceptin resulted in the specific accumulation in HER2-positive SKBR3 tumors but not normal organs (Fig. 2d). This platform does not involve covalent linkers for any component or step, including the preparation of the LPPC, drug encapsulation, and targeting antibody association. The results of Fig. 2a and Additional file 1: Figure S4 show the target molecules associated on LPPC can be changed easily and such immunocomplexes are stable enough to target antigen-positive tumors in vivo.
The cytotoxic activity of LPPC-based complex in tumor cells in vitro
The suppression of tumor growth using a LPPC-based complex in vivo
Biosafety of LPPC/Herceptin for targeting therapy
Although cationic liposome-based drug delivery particles have been revealed that they provided the better efficacy of drug delivery and therapy, cationic nanoparticles will non-specifically interact with the molecules on cell membranes of blood cells to result in agglutination, which may be dangerous to threat the life of the treated animal. The cationic LPPC particles were firstly examined their safety in this view. The results in Additional file 1: Figures S10 and S11 indicated even high dose of LPPC-PEG1500 (5 mg LPPC per mouse) did not change the body weight and cause organ damages as well as PBS treatment. Meanwhile, the results also showed ten biochemical indexes such as albumin, total protein, glucose, alanine aminotransferase, aspartate aminotransferase, creatine kinase, lactate dehydrogenase, CRE, urea and lactate were in the normal ranges for the LPPC-PEG1500 treated mice (Additional file 1: Tables S4–S6). Maximal tolerance of LPPC-PEG1500 in mice is over 30 mg per mouse for the acute toxicity. These results pointed out that PEG1500 addition significantly reduce the cytotoxic effects of LPPC and achieve the future application for drug delivery.
Pharmacokinetics of Cur/LPPC/Herceptin
Sequentially, pharmacokinetics of drug/immunocomplex was identified, and Additional file 1: Table S7 showed the half-life of curcumin in sera from the curcumin/LPPC/Herceptin was about 14 h, it is more than the half-life of non-encapsulated curcumin (less than 1 h) and similar to curcumin/LPPC. All data showed that LPPC encapsulation reduces the clearance of curcumin from the circulation and extend the duration of drug in the circulation, which may provide a better therapeutic efficacy in vivo compared with non-encapsulated curcumin.
This drug delivery platform represents a valuable technique to rapidly prepare the anti-tumor drug-encapsulated lipoplex with targeting molecules for cancer therapy by easily loading anti-tumor drug into empty LPPC and adsorbing the targeting antibody. The unloaded drugs and unbound targeting molecules were easily removed by centrifugation. This platform does not involve covalent linkers for any component or step, including the preparation of the LPPC, drug encapsulation, and targeting antibody association. However, the results show that such immunocomplexes are stable enough to target antigen-positive tumors in vivo. Figures 2, 4 and 5 clearly show that the LPPC-based drug delivery system with the adsorption of targeting molecules not only enhances the specific targeting but also displays efficient therapeutic efficacy in vivo.
Moreover, even without adsorbing targeting antibodies, LPPC encapsulation has been shown to suppress tumor growth more effectively than non-encapsulated drug by introducing higher concentrations of the drug into the tumor area. This study also showed that the adsorption of Herceptin on LPPC dramatically improves the cytotoxic effects of the drug on tumor cells compared to no adsorption of the targeting antibody (Fig. 3) by enhancing drug translocation into the tumor area (Fig. 2b). Thus, both encapsulation and the delivery of anti-tumor drug seem to yield increased therapeutic efficacy than free diffusion. Some clinically applied anti-tumor drugs fail to exert activity in vivo. If they can be modified via encapsulation or association with targeting molecules, they can provide increased therapeutic benefits for patients. The LPPC platform represents an available technique to examine the efficacy of encapsulation or targeting for clinically applied drugs in an experimental animal model.
LPPC associated with the targeting molecule Herceptin on its surface by adsorption, which is important to increase the cytotoxic effects on breast cancer cells. The key component of the adsorbing capability of LPPC is the PEI component. Without PEI incorporation into LPPC, LPPC loses the capacity to bind proteins on its surface . Therefore, the PEI component of LPPC not only plays a role in protein adsorption but also facilitates the penetration of the encapsulated drug across the cell membrane . However, PEI is highly cytotoxic due to the excess positive charges of PEI, which may be dangerous to threat the life of the treated animal. In order to reach the goal of the future application for specific drug delivery, the tissue damage of LPPC treatment must be evaluated. After treatment with LPPC mixed with PEG1500, the mice were weighted and their blood and organs were collected to evaluate the toxicity.
Unlike covenant conjugation, LPPC associated with the targeting molecule Herceptin on its surface by adsorption, which is important to increase the cytotoxic effects on breast cancer cells. In a previous publication, we showed that the key component of the adsorbing capability of LPPC is the PEI component. Without PEI incorporation into LPPC, LPPC loses the capacity to bind proteins on its surface. Compared with the liposome without PEI-coating, the results have further indicated that the PEI component of LPPC not only plays a role in protein adsorption but also facilitates the penetration of the encapsulated drug across the cell membrane. However, PEI is highly cytotoxic. This is due to the excess positive charges of PEI, which can be circumvented using PEG and blocking [25, 26]; moreover, PEG1500 blocking significantly reduces the non-specific binding and cytotoxic effects of LPPC. The pathology results show that treatments with empty LPPC did not induce organ damage in mice compared to the PBS treatments (Additional file 1: Figure S11).
Via the replacement of Herceptin with these targeting molecules, Curcumin/LPPC complexes display great potential as an improved drug delivery system for various types of cancer therapy. The increasing anti-proliferative effect of the curcumin/LPPC/Herceptin complexes may result from several possibilities: (1) LPPC-encapsulation prolongs the half-life of anti-tumor drugs (Additional file 1: Table S7), (2) Herceptin association with LPPC directs a majority of the drug-loaded LPPC to the tumor area but not healthy organs (Figs. 2c, d and 5e), and (3) the ability of the LPPC/Herceptin complexes to facilitate either drug penetration into cells or endocytosis of the drug (Fig. 2b).
Pathology results showed that the treatments with Dox/LPPC/Herceptin induced increased drug accumulation rapidly in the tumor area via vessel transport compared with LipoDox plus Herceptin. Therefore, the treatments with Dox/LPPC/Herceptin induced increased damage to vessels, thereby causing both RBC spillage and reducing the vessel numbers within the tumor (Additional file 1: Figures S7 and S8) compared with LipoDox, which may also promote enhanced tumor regression. Additionally, the accumulation of high levels of doxorubicin in tumors via Dox/LPPC/Herceptin treatment induced severe levels of tumor cell death via both apoptosis and necrosis (Additional file 1: Figure S9). Such tumor cells should not be able to successfully resist drug attack, which may be another reason for the increased tumor death following Dox/LPPC/Herceptin treatment.
With this drug delivery system, it is not only easy to encapsulate the drug and strongly adsorb targeting molecules, but it also provides a specific targeting and efficient therapeutic effect. To the best of our knowledge, no non-covalent immunoparticle has been shown to induce efficient targeting therapy in vivo as was demonstrated with the LPPC-based immunocomplex. This study demonstrates that LPPC is both an effective and available platform for the experimental investigation of specific targeting with current cancer therapies, and it is an excellent candidate for the development of targeting anti-cancer drugs in the future.
YLL, KWL and KHC designed research. YLL and CHC, YKL and KWL performed experiments and analyzed data. CJL and YLC collected the data of biochemical index of blood in animal experiment. NMT, YCC, CHL performed and analyzed histopathology. YSW and KHC performed the pharmacokinetics experiment. THH and CCW performed bio-distribution of doxorubicin. YLL and KWL wrote the manuscript. All authors read and approved the final manuscript.
The authors thank the core facility of Multiphoton and Confocal Microscope System (MCMS) and Caliper IVIS Spectrum System in NCTU, Hsinchu, Taiwan.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
All animal studies were approved by the Institutional Animal Care and Use Committee in National Chiao Tung University (NCTU-IACUC-104034).
This work was supported by Grants from Ministry of Science and Technology of Taiwan (MOST 103-2320-B-009-008, MOST 105-2314-B-341-005 and MOST105-2314-B-182A-133), from Shin Kong Wu Ho-Su Memorial Hospital Foundation (SKH-8302-103-DR-34) and from Chang-Gung Memorial Hospital Research Foundation (CMRPG2F0141-2 and CMRPG2D0091-3).
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