Potentiating antilymphoma efficacy of chemotherapy using a liposome for integration of CD20 targeting, ultra-violet irradiation polymerizing, and controlled drug delivery
- Cong Wu†1,
- Huafei Li†1, 2Email author,
- He Zhao†3,
- Weiwei Zhang1,
- Yan Chen1,
- Zhanyi Yue1,
- Qiong Lu1,
- Yuxiang Wan1,
- Xiaoyu Tian1 and
- Anmei Deng1Email author
© Wu et al.; licensee Springer. 2014
Received: 9 June 2014
Accepted: 23 August 2014
Published: 28 August 2014
Unlike most malignancies, chemotherapy but not surgery plays the most important role in treating non-Hodgkin lymphoma (NHL). Currently, liposomes have been widely used to encapsulate chemotherapeutic drugs in treating solid tumors. However, higher in vivo stability owns a much more important position for excellent antitumor efficacy in treating hematological malignancies. In this study, we finely fabricated a rituximab Fab fragment-decorated liposome based on 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC), which can form intermolecular cross-linking through the diacetylenic group by ultra-violet (UV) irradiation. Our experimental results demonstrated that after the UV irradiation, the liposomes exhibit better serum stability and slower drug release with a decreased mean diameter of approximately 285 nm. The cellular uptake of adriamycin (ADR) by this Fab-navigated liposome was about four times of free drugs. Cytotoxicity assays against CD20+ lymphoma cells showed that the half maximal (50%) inhibitory concentration (IC50) of ADR-loaded immunoliposome was only one fourth of free ADR at the same condition. In vivo studies were evaluated in lymphoma-bearing SCID mice. With the high serum stability, finely regulated structure, active targeting strategy via antigen-antibody reaction and passive targeting strategy via enhanced permeability and retention (EPR) effect, our liposome exhibits durable and potent antitumor activities both in the disseminated and localized human NHL xeno-transplant models.
Non-Hodgkin lymphoma (NHL) is a type of blood cancer, which presents not only as a solid tumor of lymphoid cells in lymph nodes and/or extranodal lymphatic organs such as spleen and bone marrow, but also as free lymphoma cells in circulating blood [1–3]. Particularly, most patients can be cured with chemotherapy and/or radiation, which revealed the important status of chemotherapy in the treatment of NHL [4–6]. Currently, while various chemotherapeutic agents are validated to be effective in the treatment of lymphoma in preclinical studies, clinical applications are often limited for their side effects to normal tissues because of the systemic administration. As a result, finding more effective strategy to maximize the curative effect while minimizing the side effects of chemotherapy against lymphoma is of great importance and urgency [7, 8].
In the past decade, nanocarriers, including liposomes, polymeric nanoparticles, micelles, nanogels etc., with an appropriate diameter of tens to hundreds of nanometers, have received widespread attention for the specific delivery of bioactive reagents in the diagnosis and treatment of cancer [7, 9–12]. Encapsulation of bioactive reagents in nanocarriers can result in significant accumulation and retention in solid tumor tissues relative to administration of drug in conventional formulations through the enhanced permeability and retention (EPR) effect, which was firstly described by Maeda and colleagues[13–17]. What's more, the drug loading nanocarriers owns high serum stability, which can contribute to long-time circulation in the blood vessels, resulting in long-lasting antitumor activities, especially for the killing of free malignant cells in circulating blood [12, 17, 18]. However, more and more laboratory researches and clinical studies have demonstrated that passive targeting strategy alone is not enough for more sufficient and efficient accumulation of drug-loading nanocarriers in some tumor types [19–21].
Following the FDA approval of anti-CD20 mAb Rituximab for CD20+ NHL treatment, monoclonal antibody (mAb)-based targeting therapy has revolutionized the treatment of malignancies for the specific antitumor activity and low cytotoxicity against normal tissues [22, 23]. In the last decade, more and more studies have confirmed that the combination of mAb-based active targeting and nanoparticle-based passive targeting can improve drug concentration in tumor tissues and tumor cells in a shorter time with greater accuracy [7, 24, 25].
In this study, we have developed an adriamycin (ADR)-loaded liposome using the diacetylenic phosphatidylcholine 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC, hereafter referred to as PC), which can form intermolecular cross-linking through the diacetylenic group to produce a conjugated polymer within the hydrocarbon region of the bilayer by ultra-violet (UV) irradiation (Additional file 1: Figure S1) [26, 27]. For the sake of active targeting, the Fab fragments of rituximab were conjugated onto the liposomal surface. Our experimental results demonstrate that this well-modified liposome, which owns good serum stability and prolonged circulation time, can accumulate in the tumor tissues and malignant cells with high specificity and sufficient amount, which can bring out exceptional excellent and durable therapeutic efficacy against CD20-positive lymphomas.
Cell lines and materials
Two human B cell lymphoma cell lines, Raji and Daudi, were obtained from the American Type Culture Collection (ATCC). Cells were propagated and maintained in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum (FBS, GIBCO, Invitrogen, Carlsbad, CA, USA) in a controlled atmosphere incubator at 37°C with 5% CO2. The DC8,9PC and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (Mal-PEG) were purchased from Avanti Polar Lipids (Williamsport, PA, USA). The anti-CD20 antibodies rituximab was purchased from Roche (Basel, Switzerland).
Fabrication of Fab fragment-conjugated liposome (Figure 1)
Formation of drug-loaded liposomes
Total lipids mixtures of 2 mg DC8,9PC and 0.25 mg Mal-PEG were dissolved in 500 μL mixed solvent of chloroform and methyl alcohol with the volume ratio at 1:1. Then, the solvent was evaporated under vortex and flashed with nitrogen to obtain the lipid film, followed by washing-out with 2 mL of ADR (doxorubicin HCl, Melonepharma CO. LTD., Dalian, China) solution (0.5 mg/mL in PBS) to obtain ADR-loaded multilamellar vesicles . The collected liposome solution was dialyzed against PBS using a membrane (molecular weight cutoff 3 kDa) at 4°C for 12 h to remove uncombined ADR resulting in the ADR-loaded liposome stocking solutions.
Thiolation of mAbs
The Fab fragment of rituximab was prepared as reported previously . Briefly, 10 mg/mL Rituximab was incubated with 0.25 mg/mL de pepsin at 37°C overnight following with a centrifugation and dialysis against TBS (145 mM NaCl and 10 mM Tris, pH 7.5) for 18 h. The Staph A-Sepharose column (Pharmacia, Kalamazoo, MI, USA) was used to remove the undigested mAbs at pH 8.0 resulting in the rituximab F(ab)2. The obtained F(ab)2 was further purified by the Sephadex G-150 column (Pharmacia, MI, USA) which was pre-equilibrated by the buffer 1 (0.1 M NaCl, 0.1 M borate, 0.05 M citrate and 2 mM EDTA, pH 5.5). Such F(ab)2 solution was concentrated to 10 mg/mL and further digested by the enzyme papain. The Fab fragment solution was purified by the same procedure as mentioned above resulting in the single Fab fragment stocking solution storing at 4°C.
To activate the Fab fragments of rituximab for reactivity toward the maleimide, the above stocking solutions were incubated with 2-iminothiolane (2-IT, Sigma-Aldrich, St. Louis, MO, USA) with a mass ratio of 1:0.15 (Fab/2-IT) at room temperature for 2 h under a gentle shake. Unreacted 2-IT was removed by dialysis. The bovine serum albumin (BSA) ~ SH was produced in the same way. The resulting reactive Fab ~ SH and BSA ~ SH were stored at 4°C for future usage .
Fabrication of rituximab Fab-conjugated liposome
The Fab fragment-conjugated liposome was prepared by coupling the reactive Fab ~ SH onto the liposomal surface via the reaction between the ~ SH and Mal-group at 4°C and N2 environment overnight; the un-conjugated Fabs were removed by dialysis. The BSA-conjugated liposome was fabricated in the same way. For UV irradiation, pure liposome solutions were exposed to 20 irradiation cycles at 4°C, with a 254-nm UV light dose of 360 mJ/cm2 per cycle using a Stratalinker-UV 1800 . The concentration of Fab fragments in the liposome solution was quantified by measuring the A260/A280 using Nano VueTM (GE Healthcare, Wilmington, MA, USA).
Characterization of Fab fragment-conjugated liposome
The hydrodynamic diameter and size distribution were determined by ZetaSizer (Nano-ZS, Malvern Instruments, Worcestershire, UK) equipped with a HeeNe laser (633 nm) at the scattering angle 173°. To prepare stained specimens for TEM (H-7000 Electron Microscope, Hitachi, Tokyo, Japan) experiments, about 5 μL liposome solution was dropped on 200-mesh Formvar-free carbon-coated copper grids (Ted Pella Type-A; nominal carbon thickness 2 to 3 nm). After the water evaporating by exposing to air at room temperature, the sample was inversely covered on a small drop of hydrodated phosphotungstate (PTA) solution with a mass fraction of 2%. The conventional TEM images were obtained at 100 kV.
Weight-average molecular weight analysis by SLS
where K = [4π2n2(dn/dC P )2]/NAVλ4 is optical contrast, with n being the refractive index of solvent, C p the liposomal concentration, dn/dC p the refractive index increment against C p determined by a double beam differential refraction meter (DMR-1021) (Otsuka Electronics, Tokyo, Japan), λ the incident wavelength, and NAV the Avogadro's number. R(q) is the Rayleigh ratio at a specific measurement angle. By measuring R(q) for a set of θ and C p , values of M w and A2 were estimated from typical Zimm plots.
ADR releasing profile
Serum stability evaluation by DLS
For evaluating the effect of UV irradiation on the liposomal stability, a bovine serum albumin (BSA) solution in RPMI 1640 with a concentration of 50% (m/v) was used as an in vitro serum model to mimic the in vivo status. Then, the irradiation (irrad) and non-irrad liposome solutions were separately mixed with the resulting serum model at 37°C for 24 h. The dynamic light scattering (DLS) was used to measure the size and size distribution profile of BSA/liposome mixture at 0 and 24 h, respectively.
Cellular uptake and internalization assays
Raji and Daudi cells were seeded into a 48-well microplate (1 × 105 cells) and incubated with 1 μg/mL free ADR, ADR-loaded liposomes decorated with Fab fragments (PC-ADR-Fab), or BSA (PC-ADR-BSA) in cell culture medium containing 1% (v/v) antibiotics at 37°C for 4 h. Cells incubated with culture medium were used as a negative control. After washing with PBS for twice, a FACScan Flow Cytometer (Becton Dickinson, San Jose, CA, USA) was used to assess the cellular uptake of ADR or ADR-loaded liposomes by detecting the mean fluorescence intensity (MFI) of FL-2 (ADR fluorescence). Additionally, each sample was also visualized using an inverse fluorescent microscopy.
In vitro cytotoxicity assay
where Absample, Abcontrol, and Abblank are the absorbance values of each sample, the cells cultured in culture medium without any additional substances, and the culture medium without cells in wells, respectively.
Healthy female SCID mice aged about 4 weeks were purchased from Shanghai Experimental Animal Center of Chinese Academic of Sciences (Shanghai, China), housed in specific pathogen-free conditions, and treated in accordance with guidelines of the Committee on Animals of Changhai Hospital affiliated to the Second Military Medical University (Shanghai China).
Pharmacokinetics and in vivo distribution analysis
The pharmacokinetics (PK) and in vivo distribution analysis was done following Joseph M. Tuscano's study with minor revisions . Briefly, Daudi cells (1 × 107) were inoculated subcutaneously into the right flank of 6-week-old SCID mice. For PK assays, when tumors reached about 50 to 60 mm3 in volume (approximately 14 days), mice were randomly administrated tail vein injection of free ADR, non-irrad or irrad ADR-containing immunoliposomes at a dosage of 5 mg ADR/kg (n = 3 mice per treatment). Then, 10 μL of blood were collected through tail vein nicking from each mouse at 5, 15, and 30 min and 1, 2, 4, 6, 8, 12, 24, and 48 h after treatment, respectively. Samples were immediately diluted into 250 μL of 0.5 mmol/L EDTA-PBS, followed by a centrifugation (300 g × 5 min). Plasma was collected and ADR was extracted by acidified isopropanol (75 mmol/L hydrochloric acid in 90% isopropanol) at 4°C for 20 h. The ADR concentrations were measured by UV at 480 nm and expressed as micrograms per milliliter (ADR/blood plasma). The data were analyzed by the PK solver software . For biodistribution assays, tumor-bearing mice were randomly administrated tail vein injection of free ADR, PC-ADR-BSA, or PC-ADR-Fab at a dosage of 5 mg ADR/kg (n = 3 mice per treatment). Mice were sacrificed 24 h after treatment; part of tumor, heart, liver, spleen, kidneys, and lungs were removed, washed, and weighed; and single-cell suspensions were made. ADR was extracted from cells by the abovementioned acidified isopropanol for 20 h at 4°C. The ADR concentrations were determined as described above and expressed as micrograms per gram (ADR/tissue). What's more, part of the tumor tissues were collected and subjected to frozen sections, which were detected by a confocal microscrope (Zeiss, Oberkochen, Germany).
In vivo antitumor activity assessment in disseminated human NHL xeno-transplant models
Six-week-old SCID mice were injected via the tail vein with 5 × 106 Daudi cells in 100 μL PBS. Then, the inoculated mice were randomly assigned to 4 groups with 10 each for the treatment of PBS, free ADR, PC-ADR-BSA, and PC-ADR-Fab (with an equivalent amount of 5 mg/kg ADR) via the tail vein weekly for 3 times after 48 h. Post-operation monitoring was exercised at least once a day until natural death in a range of 120 days. Survival curves were plotted with the Kaplan-Meier method and compared by using a log-rank test [33, 34].
In vivo antitumor activity assessment in localized human NHL xeno-transplant models
where length and width refers to the longest and the shortest diameters of tumors, respectively.
Data were expressed as the means ± standard deviation (SD). Statistical analysis was performed by Student's t test or one way ANOVA to identify significant differences unless otherwise indicated. Differences were considered significant at a P value of <0.05.
Characterization of the liposome
Fab fragment loading
The number of Fab fragments per liposome was estimated on the basis of Kozlowska's ideas according to the following equation :
Physicochemical parameters of ADR-loaded immunoliposomes
R h (nm)
M w (g/mol)
1.22 × 107
3.1 × 10-9
The number of Fab fragments (24 kDa) per milliliter calculated in the same way was 2.2 × 10-9 mol [52.2 μg/(2.4 × 104 g/mol)] or 1.3 × 1015. Hence we can estimate that there are on average ~31.3 Fab fragments per liposome (1.3 × 1015 Fab fragments/4.15 × 1013 liposomes), which is also shown in Table 1.
Drug loading and releasing properties
It was well expected that our liposome could be an excellent drug carrier which benefits from the stable structure following by self-assembling and UV irradiation functions. For the validation of this expectation, we firstly evaluated the ADR loading content (LC) of our liposomes according to the following function: . The results revealed a relative high LC of 16.27% with our immunoliposomes. Besides, the amount of ADR per liposome was estimated to be 3.1 × 10-9 ng (Table 1), which was calculated according to the following equation:
Low cytotoxicity of liposomes
For the determination of the cytotoxicity, different concentrations of empty liposomes decorated by BSA (PC-BSA) and rituximab Fab fragments (PC-Fab) were incubating with Raji cells at 37°C for 48 h following by a CCK-8 detection. As illustrated in Figure 2D, both the PC-BSA and PC-Fab showed low cytotoxicity to Raji cells in concentrations of up to 32 μg/mL. It is worth mentioning that the cell viability of PC-Fab-incubated cells had a little decrease compared with PC-BSA-incubated cells, which may be related with the weak tumor suppression effect of rituximab Fab fragments.
Serum stability evaluation
Intracellular uptake of liposomes
In vitro cytotoxicity assays
Pharmacokinetics of ADR-containing liposomes in tumor bearing SCID mice
Tumor bearing nude mice serum pharmacokinetic parameters comparing free and liposomal ADRs ( n = 3)
0.20 ± 0.02
0.19 ± 0.04
0.21 ± 0.05
0.98 ± 0.19
3.89 ± 0.79
1.57 ± 1.31
9.56 ± 4.06
21.13 ± 1.50
34.53 ± 2.63
30.96 ± 5.86
8.82 ± 4.54
6.63 ± 3.74
50.45 ± 5.54
54.13 ± 4.34
53.04 ± 5.68
(μg/mL) · h
79.97 ± 11.36
447.19 ± 54.19
713.49 ± 120.51
6.37 ± 2.15
27.54 ± 1.53
48.58 ± 4.67
In vivo distribution and tumor accumulation assays
In vivo antitumor activity assessment
For the evaluation of in vivo antitumor activities, both the disseminated and localized human NHL xeno-transplant models were set up. In the localized model, Daudi cells were inoculated subcutaneously in the right flank of SCID mice. When the tumors reached about 50-60 mm3 in volume, mice were randomly treated with free ADR, PC-ADR-BSA and PC-ADR-Fab with an equivalent ADR amount of 5 mg/kg [25, 38]. The mice were treated once a week for SCID mice based on previous study and our preliminary experimental results . The tumor volume was recorded and illustrated in Figure 6C. Our results indicated that mice treated with PC-ADR-Fab and PC-ADR-BSA demonstrated a remarkable decrease in tumor burden compared with free ADR and control treatment as measured by tumor volume. Otherwise, PC-ADR-Fab exhibit a more excellent antitumor ability comparing with PC-ADR-BSA, with 2/4 mice of complete remission (CR) indicated by no measurable mass.
The excellent antitumor activity of our liposome is validated using a disseminated model, in which Daudi cells were transplanted intravenously into SCID mice via tail vein. After 48 h, these mice were randomly administered injections of PBS, free ADR, PC-ADR-BSA, and PC-ADR-Fab for three times once a week. Survival curves were plotted with the Kaplan-Meier method and were compared by using a log-rank test [33, 34]. As illustrated in Figure 6D, ADR-loaded liposome (PC-ADR-BSA and PC-ADR-Fab) treatment significantly prolonged the survival of tumor-bearing mice compared to free ADR and PBS control treatment (*p < 0.05). As our expectation, comparing with PC-ADR-BSA treatment, the administration of PC-ADR-Fab led to significant prolongation of graft survival days (*p < 0.05), with a CR percentage of 4/10 indicated by long-term survival (>120 days post-treatment).
NHL presents not only as a solid tumor of lymphoid cells in lymph nodes and/or extranodal lymphatic organs, but also as free lymphoma cells in circulating blood [1–3]. Unlike most other malignancies, chemotherapy but not surgery plays the most important role in curing NHL [4–6]. Currently, more and more studies are focusing on finding out novel drug delivery system for treating solid tumors [7, 11, 17, 25]. However, for the elimination of free malignant cells in circulating blood, high serum stability and specificity to tumor cells are of great importance.
In this study, we have successfully fabricated a rituximab Fab-conjugated liposome based on PC, of which the well-defined spherical morphology was observed under TEM. Because PC is a kind of diacetylenic lipids, which can form intermolecular cross-linking through the diacetylenic group by UV irradiation to form chains of covalently linked lipids in the liposomal bilayers (Additional file 1: Figure S1) , this covalently union between lipid chains leads to a relatively more compact structure; thus, an important impact on the stability of the polymerized drug delivery system can be obtained. This enhanced serum stability can result in longer-time circulation and slower clearance of encapsulated drugs in vivo. Further experimental results revealed a favorable biological compatibility of the liposome. All the abovementioned properties are of vital importance for an ideal drug delivery system in eliminating malignant lymphoma cells, especially those in the peripheral blood.
In order to determine the antitumor activities, we took two lymphoma cell lines, Raji and Daudi, as study targets. The experimental results demonstrated that the in vitro cytotoxicity of ADR-loaded targeting liposome (PC-ADR-Fab) was significantly promoted due to the enhancement of drug uptaking compared with free drugs and ADR-loaded non-targeting liposome. The result may be ascribed to the following two reasons. Firstly, previous studies have proven that nanoparticles are taken up by cells via clathrin and/or caveoli-mediated endocytosis unlike small molecule drugs, which were taken up by passive diffusion [40, 41]. Thus, most nanoparticles can obviously enhance the intracellular uptake of chemotherapeutic agents, which was confirmed by previous studies and recognized as an important advantage of nanosized drug delivery system [25, 42, 43]. Secondly, the intracellular uptake could be further improved by the Fab fragments of rituximab based on the active targeting strategy by antigen-antibody identification and combination.
In vivo experimental results indicated that the immunoliposomes are selectively accumulated in tumor tissues, while the administration of free drugs resulted in high concentration of ADR in either normal or malignant tissues with no specificity. This remarkable discrepancy can significantly improve the bioavailability and reduce the detrimental cytotoxicity of chemotherapeutic agents. The in vivo antitumor experiments carried out both in the localized and disseminated human NHL xeno-transplant models suggest that our immunoliposome was significantly more effective than either free ADR or non-targeting liposomal ADR in inhibiting primary tumor growth and prolonging the graft survival. What's more, our immunoliposome still showed great advantage in tumor suppressing efficacy when compared with other drug delivery systems. For example, comparing with the anti-CD30 antibody-conjugated liposomal doxorubicin constructed by Ommoleila Molavi et al., the treatment of which can respectively decrease the tumor burden to approximately 1/7 and approximately 1/2 in comparison with PBS and free ADR treatment ; our immunoliposome can remarkably decrease the tumor burden to approximately 1/14 and approximately 1/4, respectively. In our opinion, this exceptional excellent in vivo antilymphoma activity of the ADR-loaded Fab fragment-decorated liposome is the cooperative action of the following effects: (1) enhanced intracellular uptake due to effective endocytosis based on well-defined liposomal structure and size distribution; (2) enhanced serum stability and controlled drug release (as a result of UV irradiation polymerizing) can contribute to long circulation time and durable antilymphoma activity; (3) enhanced tumor accumulation and retention in vivo through dual targeting function, passive targeting through EPR effects and active targeting through antigen-antibody reaction.
In this study, we have identified a novel liposomal drug delivery system, PC-Fab, for improved chemotherapy of CD20-positive NHL. The in vitro and in vivo experimental results clearly suggested that this Fab fragment-decorated liposome can be a promising weapon in combating NHL, which deserves further investigation for clinical application.
This research was supported by grants from 973 Foundation (2013CB531606), National Science Foundation of China (81371786), Shanghai Municipal Commission for Science and Technology (11JC1410902), Youth fund of the Second Military Medical University (2013QN14), and Wujieping Grant (320.6750.13147).
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