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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Gene therapy shows promising application in cancer therapy, but the lack of an ideal gene delivery system is still a tough challenge for cancer gene therapy. Previously, we prepared a novel cationic nanogel, heparin-polyethylenimine (HPEI), which had potential application in gene delivery. In the present study, we constructed a plasmid with high expression efficiency of interleukin-15 (IL15) and investigated the effects HPEI–plasmid IL15 (HPEI–pIL15) complexes on the distribution level of the lung. We then evaluated the anticancer effect of HPEI–pIL15 complexes on lung metastases of B16-F10 melanoma and CT26 colon carcinoma. These results demonstrated that intravenous injection of the HPEI–pIL15 complex exhibited the highest plasmid distribution level in the lung compared with that of PEI2K–pIL15 and PEI25K–pIL15, and mice treated with HPEI–pIL15 had a lower tumor metastasis index compared with other treatment groups. Moreover, the number of natural killer cells, which were intermingled among the tumor cells, and the level of tumor necrosis factor-α and interferon-γ in the serum also increased in the pIL15-treated mice. Furthermore, the cytotoxic activity of spleen cells also increased significantly in the HPEI–pIL15 group. In addition, induction of apoptosis and inhibition of cell proliferation in lung tumor foci in the HPEI–pIL15 group was observed. Taken together, treating lung metastasis cancer with the HPEI nanogels delivered by plasmid IL15 might be a new and interesting cancer gene therapy protocol. (Cancer Sci 2011; 102: 1403–1409)

Lung metastasis is the most common cause of cancer death in both men and women. Investigators have done a lot to improve its prognosis and the development of new modalities of treatment. Gene therapy is a promising and powerful method; currently, its potential in cancer treatment is widely recognized. However, the major limitation of gene therapy of the lung is the low efficiency of gene transfer and the technical difficulties of regimen delivery.(1) Delivery vectors play an important role in gene therapy because the plasmid DNA must be protected from damage and its entry into the cell must be facilitated. Some polymers and cationic lipids have shown high gene transfection efficiency in the lung for DNA encapsulated.(2) The cationic polymer polyethylenimine (PEI) has the ability to protect DNA from undesirable degradation during the transfection process and is one of the most effective non-viral gene vectors.(3–5) However, PEI also has a shortcoming: the increase in transfection efficiency is accompanied by an increase in cytotoxicity, and both efficiency and cytotoxicity increase as its molecular weight increases. To address this challenge, we used heparin to couple PEI together, forming HPEI nanogels; these nanogels had high transfection efficiency and low toxicity, showing promising application as a non-viral gene delivery system.(6)

Interleukin-15 (IL15) is a cytokine first identified in the supernatant of the monkey epithelial cell line CV-1/EBNA and has a similar structure to interleukin-12 (IL12).(7) Like IL2, IL15 binds to and signals through the IL2/IL15 beta chain (CD122) and the common gamma chain (gamma-C, CD132).(8,9) Interleukin-15 exerts its antitumoral activity through activation of the innate and acquired immune system. Interleukin-15 plays pivotal roles in the development, survival and activation of natural killer (NK) cell lineages(10) and the growth promotion of T cells(11,12) and B cells.(13) It can also promote the functional maturation of dendritic cells and macrophages,(14) and induce production of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ).(15,16) Gene delivery of immunostimulatory cytokines, such as IL15 or IL12, has been shown to be highly effective in eradicating tumors in different animal models including lung metastasis of cancer.(17–21)

In this study, we found that HPEI nanogels can more efficiently deliver pIL15 to the lung compared with PEI2K and PEI25K. Moreover, we investigated the potential effects of HPEI–pIL15 on the inhibition of lung metastases of B16-F10 malignant melanoma and CT26 colon carcinoma in a murine model. The results suggested that pIL15 delivered by HPEI nanogels had potential application in gene therapy for lung metastasis of cancer.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Animals and cells.  C57BL/6 and BALB/c female mice, 4–5 weeks old, were obtained from the Laboratory Animal Center of Sichuan University. All animals were handled in strict accordance with good animal practice. CT26, B16-F10 and CHO-K1 cells were obtained from American Type Culture Collection (Manassas, VA, USA). CTLL-2 cells were obtained from Cell Resource Center, Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences (Shanghai, China).

Generation of pcDNA3.1–IL15.  An IL2 signal peptide (GenBank Accession No. NM000586.3) was inserted upstream of the mature IL15 gene by multi-step PCR. The template for the PCR reactions was the IL15-encoding plasmid pORF–IL15 (InvivoGen, San Diego, CA, USA). The isolated PCR product was cloned into the pcDNA3.1 (+) expression vector.

Preparation of HPEI–pDNA complexes.  Heparin (Mw = 4000–6000 kDa) and polyethyleneimine (Mw = 2000 kDa, PEI2K) were purchased from Fluka (Milwaukee, WI, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. The heparin-conjugated polyethylenimine was prepared as previously described.(6) Briefly, 50 mg of heparin was dissolved in 100 mL of 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution (0.05 M). To activate the carboxylic acid groups of heparin, 20 mg of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and 30 mg of N-hydroxysuccinimide (NHS) were added into the above solution. Two hours later, this solution was dropped into PEI2K solution (20 mL, 7.5 mg/mL) while consistently stirring. The reaction was carried out at room temperature overnight. Finally, the resultant HPEI nanogels were dialyzed in distilled water, and pure HPEI nanogels were obtained. Heparin-polyethylenimine containing the pcDNA3.1–IL15 plasmid (HPEI:pIL15 at a PEI nitrogen:DNA phosphate ratio of 8:1) was prepared and then mixed by vortexing and incubated for 15 min at room temperature.

Western blot analysis and in vitro bioactivity assay.  B16 and CT26 cells grown in tissue culture plates (3 × 105 cells/well in a six-well plate) were transfected with HPEI–pDNA complexes for 6 h. After 48 h, the supernatants and cell layer were collected. Protein of the supernatants and transfected cells were detected by polyclonal goat monoclonal anti-IL15 antibody (1:1000; R&D Systems, Minneapolis, MN, USA).

CHO-K1 cells were transfected with HPEI–pIL15 complexes for 6 h. After 48 h the transfection media was collected. Cytokine activity of IL15 in the transfection media was determined by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; Sigma, St. Louis, MO, USA) dye reduction assay on CTLL-2 cells as previously described.(20,22)

Lung biodistribution experiments.  HPEI–pIL15, PEI2K–pIL15 and PEI25K–pIL15 complexes (30 μg plasmid, nitrogen/phosphorus [N/P] ratio of 8:1) were injected in a final volume of 0.5 mL of normal saline into the tail vein of C57BL/6 mice. Mice were killed 24 h after injection. The lungs were removed, washed with saline, blotted dry and weighed. Total genomic DNA was extracted from the lungs. Amplification was detected on an iQ5.0 instrument (Bio-Rad, Hercules, CA, USA) using a SYBR Green qPCR Kit (TaKaRa, Dalian, China). The level of distribution of the target plasmid was normalized to that of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) in the same sample.

Murine tumor models and treatment.  A B16-F10 and CT26 lung metastasis model was induced by tail vein injection of 2 × 105 cells in C57BL/6 mice and BALB/c mice on day 0. Tumor-bearing mice were then randomly assigned to one of the following four groups (n = 9 mice per group): (i) mice treated with normal saline (NS); (ii) mice treated with empty HPEI; (iii) mice treated with HPEI/pcDNA3.1-Null (HPEI–pNull); and (iv) mice treated with HPEI–pcDNA3.1-IL15 (HPEI–pIL15).

HPEI–pDNA complexes were prepared as above and injected intravenously 3 days after tumor cell injection. The HPEI–pDNA complex containing 5 μg plasmid or the same dose of HPEI was given every 2 days for a total of six doses (on days 3, 6, 10, 13, 16 and 19). The animals were killed 24 h after the last dose and the lungs were harvested and weighed. The therapeutic effect of systemic pIL15 treatment was determined by counting the number of metastatic tumors in each lung under a dissecting microscope without knowledge of the treatment groups. As most of the lungs in the control groups had numerous uncountable foci, the lungs were graded based on a scale of 1–5 as previously reported.(23)

Spleen cell-mediated cyotoxicity assay.  The animals were killed 24 h following the last dose and the splenocytes were collected. Spleen cells were added to the target cells (B16 or CT26) at effector to target (E:T) ratios of 10:1, 20:1 and 40:1. After 6 h, the cytotoxic activity of splenocytes was measured by a lactate dehydrogenase (LDH) release assay kit (Promega, Fitchburg, WI, USA) on 5 × 103 target cells per well. Experiments were performed according to the manufacturer’s protocol.

Quantification of serum TNF-α and IFN-γ.  Sera were collected before the mice were killed and tested in duplicate for the production of TNF-α and IFN-γ using mouse TNF-α and IFN-γ ELISA kit (Neobioscience, Shenzhen, China). All reagents were used according to the manufacturer’s protocol. Absorbance was read at 450 nm using an ELISA multi-well spectrophotometer (Biotek Instruments Inc., Winooski, VT, USA).

Immunohistochemistry and TUNEL assay.  Mouse anti-human proliferating cell nuclear antigen (PCNA) antibody (1:500; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was applied for detecting tumor cell proliferation on the paraffin section. The NK cells were detected using Anti-asialo GM1 antibody (1:1000; Wako, Osaka, Japan) on lung frozen tissue sections. TUNEL detection analysis of apoptotic cells in tumor tissue was measured using the TUNEL Apoptosis Detection kit (Upstate LabChem Inc., Lake Placid, NY, USA) following the manufacturer’s directions.

Toxicity assessment.  Possible side-effects were observed through weight, appetite, diarrhea, life span and behavior until the mice were killed. Organs such as the heart, liver, spleen, lung and kidney were collected and made into 4-μm sections, which were stained with hematoxylin and eosin (HE) and observed by two pathologists in a blinded manner under a microscope.

Statistical analysis.  Data are expressed as the mean ± SD. Statistical analysis was performed by Student’s test for comparing two groups and by anova for multiple group comparisons. All statistical tests were two sided. SPSS 16.0 (IBM, Chicago, IL, USA) was used for all statistical analyses.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Preparation of HPEI–pIL15 complexes.  To develop an efficient and safe gene vector and carrier, we constructed a plasmid with high expression of IL15 protein and prepared a biodegradable cationic nanogel: HPEI nanogels. A schematic diagram of the pcDNA3.1–IL15 constructs is presented in Figure 1(A). It contained a CMV promoter, IL2 signal peptide and IL15 cDNA sequences. Heparin-polyethylenimine nanogel was also synthesized in a simple improved method (Fig. 1B). Heparin is a biodegradable negative polysaccharide with many carboxylic groups in its molecular structure. Polyethylenimine is a cationic polymer with many primary amine groups in its molecular structure. Therefore, the reaction would occur between heparin and PEI when EDC and NHS were added. Catalyzed by EDC/NHS, a reaction between an amino group and a carboxyl group occured, forming amine groups. This led to one heparin molecule reacting with several PEI molecules, and also cross-linkage between heparin and PEI occurred; thus, through an amide bond heparin conjugated PEI molecules, forming a HPEI nanogel. The prepared HPEI nanogels were characterized in detail, and the results are presented in a previous report.(6) The size distribution spectrum of HPEI nanogels indicated that HPEI nanogels were monodisperse (polydispersion index [PDI] = 0.157) and the mean particle size was 75 ± 6.6 nm. The zeta potential spectrum of HPEI nanogels presented that these HPEI nanogels were cationic and had the zeta potential of +27 ± 0.71 mV. Moreover, these HPEI nanogels were likely to have high water absorption, as dry HPEI nanogels (approximately 25 nm) can absorb water and swell to become nanogels with a size of approximately 75 nm.

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Figure 1.  Preparation of heparin-polyethylenimine (HPEI)–plasmid interleukin-15 (pIL15) complexes. (A) Schematic diagram of pcDNA3.1–IL15 constructs containing interleukin-2 (IL2) signal peptide and IL15 cDNA sequences. (B) Preparation scheme of heparin-polyethylenimine (PEI) nanogel.

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In vitro gene transfer and expression of IL15.  The transfection efficiency of HPEI–pIL15 complexes on the B16-F10 and CT26 cell lines was evaluated in vitro. After transfection with the pcDNA3.1–IL15 plasmid, expression of secreted IL15 was detected by western blot analysis in serum-free medium and tumor cells transfected with pcDNA3.1-Null plasmid as a control. Figure 2(A) shows the detection of IL15 in the media of pcDNA3.1–IL15 transfected cells but not in that of pcDNA3.1-Null transfected cells.

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Figure 2. In vitro detection of interleukin-15 (IL15). (A) Production of hIL15. B16-F10 and CT26 cells were transfected with heparin-polyethylenimine (HPEI)/pcDNA3.1-Null (pNull) or HPEI–plasmid IL15 (pIL15). After 48 h, media and cells were collected and assayed for hIL15 using western blot. (B) Survival rate of CTLL-2 cells. Media obtained from HPEI–pNull or HPEI–pIL15 transfected CHO-K1 cells was added to CTLL-2 cell culture. After 72 h, the survival rate of CTLL-2 cells was determined by MTT assay. Data were obtained from three independent triplicate experiments and presented as mean ± SD (*P < 0.01).

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We also analyzed the bioactivity of the secreted IL15 in vitro. Observation of the CTLL-2 cell survival rate showed that CTLL-2 cells could grow with the media obtained from pcDNA3.1–IL15 transfected CHO-K1 cells. However, media obtained from pcDNA3.1-Null transfected CHO-K1 cells resulted in no effect on cell growth of CTLL-2 cells (Fig. 2B).

Pulmonary distribution of HPEI.  The distribution and retention patterns of plasmid DNA in the lung was a very important factor for the successful treatment of lung cancer. At 24 h following intravenous injection of pcDNA3.1–IL15 plasmid to various PEI complexes at N/P ratio 8, we evaluated the different distribution level of plasmid DNA in the lungs using real-time PCR. The relative lung distribution level of HPEI–pIL15 is presented in Figure 3. The polyplexes of HPEI exhibited the highest plasmid distribution in the lung while the PEI2K group showed the lowest detectable plasmid. In addition, the plasmid levels in the lung were 1.7-fold and fivefold higher in the group treated with HPEI compared with the groups given PEI25K and PEI2K at the same N/P ratio, respectively.

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Figure 3.  Lung distribution level of plasmid interleukin-15 (pIL15) after intravenous delivery in various polyethylenimine (PEI) complexes. Thirty micrograms of pcDNA3.1–IL15 was injected into the tail vein of mice and the nitrogen/phosphorus (N/P) ratio was 8:1. The mice were killed 24 h after bolus injection. Total genomic DNA was extracted from the lung and pcDNA3.1–IL15 levels were quantified by real-time PCR assay. The results are expressed as mean ± SD (n = 3 mice, *P < 0.05).

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Inhibition of B16-F10 and CT26 lung metastasis by intravenously delivery of HPEI–pIL15 complexes.  In the model of pulmonary metastasis of melanoma and colon cancer, BALB/c mice received 2 × 105 B16-F10 and CT26 cells through the tail vein, respectively. Three days later, the mice were divided into four different groups (n = 9) and treated with NS, HPEI, HPEI-pNull and HPEI–pIL15. The mice treated with HPEI–pIL15 in both B16-F10 and CT26 tumor models had a very low tumor metastasis index (< 0.05 compared with all other groups), whereas the majority of mice treated with NS, HPEI alone or HPEI-pNull had a higher tumor index (Fig. 4A–C). The lung weights also showed a significant difference (< 0.05) between the HPEI–pIL15-treated group and all the control groups (Fig. 4D,E). Furthermore, some mice treated with NS, HPEI or HPEI–pNull complexes in the B16-F10 and CT26 tumor models had tumors in the kidney or abdominal lymph nodes, but none of the mice treated with HPEI–pIL15 had any metastasis to extrapulmonary tissue (data not show).

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Figure 4.  Inhibition of B16-F10 and CT26 lung metastasis by heparin-polyethylenimine (HPEI)–plasmid interleukin-15 (pIL15) intravenous delivery. Mice were injected via lateral tail vein with B16-F10 and CT26 cells on day 0. The mice were then treated with normal saline (NS), HPEI alone, HPEI–pcDNA3.1-Null (pNull) or HPEI–pIL15 complexes every 2 days for a total of six doses starting on day 3 (n = 9 mice per group). (A) Representative lungs of B16-F10 and CT26 models from NS, HPEI and HPEI–pNull control groups and HPEI–pIL15-treated mice. (B,C) Tumor index and lung weights of mice from different groups of the B16-F10 tumor model. (D,E) Tumor index and lung weights of mice from different groups of the CT26 tumor model. Data are representative of at least two separate experiments. Bars, means ± SD (*P < 0.05).

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Induction of antitumor cytotoxic activity and TNF-α and IFN-γ production.  To further assess if HPEI–pIL15 could induce spleen cell-mediated cyotoxicity, the splenocytes separated from HPEI–pIL15-treated mice were detected ex vivo by a LDH release assay. From Figure 5, it is suggested that spleen cells isolated from the mice of control groups did not lyse B16-F10 and CT26 cells. However, antitumor cytotoxic activity of spleen cells induced in the HPEI–pIL15 group in the B16-F10 and CT26 tumor models were 25% and 32%, 18% and 25%, 15% and 15% at the E/T ratio of 40:1, 20:1 and 10:1, respectively (Fig. 5A,B).

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Figure 5.  Splenic cell-mediated cytotoxicity, natural killer (NK) cell recruitment and tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) expression levels. The splenocytes were separated 24 h after the last dose and incubated with B16-F10 (A) and CT26 (B) at various effector-to-target (E:T) ratios. (C,D) TNF-α and IFN-γ induction in the serum of C57BL/6 and BALB/c mice after treatment with heparin-polyethylenimine (HPEI)–plasmid interleukin-15 (pIL15). Bars, means ± SD (*P < 0.05, n = 3 mice). (E) Different numbers of NK cells are intermingled among the HPEI–IL15-treated cells and normal saline (NS)-, HPEI- or HPEI-Null-treated cells.

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We next evaluated the changes in cytokine levels in an in vivo experiment. The serum from B16–pIL15 and CT26–pIL15 animals contained significantly higher levels of TNF-α and IFN-γ (1353.3 and 900 pg/mL, and 133 and 170 pg/mL, respectively) compared with control groups (Fig. 5C,D). This result suggested that administration of HPEI–pIL15 was able to increase the in vivo production of the inflammatory cytokines TNF-α and IFN-γ.

We also observed impressive NK cell recruitment in pIL15-treated tumors via an immunofluorescence study. As shown in Figure 5(E), many NK cells were intermingled among B16–pIL15 and CT26–pIL15 cells. However, these cells were scarcely present in B16–HPEI, B16–pNull, CT26–HPEI and CT26–pNull tumor.

Inhibition of cell proliferation and induction of apoptosis in lung tumor foci.  Usually, tumor growth is often considered as a destroying of the balance between apoptosis and proliferation. To address if there were some phenotypic changes of tumor tissues, we first examined the percentage of apoptotic cells in tumor tissues. As shown in Figure 6(A), significant increases of TUNEL-positive nuclei were found in the HPEI–pIL15-treated group compared with that in tumor tissues of mice treated with NS, HPEI and pNull. We further determined the effects of HPEI–pIL15 on tumor cell proliferation using PCNA staining. The expression of PCNA was dramatically reduced in the HPEI–pIL15-treated group compared with other groups (Fig. 6B). Our data showed the percentage of PCNA-positive cells in the B16-F10 and CT26 tumor models reached 82 ± 5.3% and 85 ± 3.4%, 77 ± 4.3% and 83 ± 6.3%, 72 ± 4.1% and 76 ± 4.5% in the NS, HPEI and HPEI–pNull groups, respectively, whereas corresponding values in the HPEI–pIL15-treated group reached only 46 ± 4% and 31 ± 2.8%, respectively. Therefore, expression of IL15 in the lung tumor foci could inhibit cell proliferation and increase apoptosis of tumor cells in vivo, which was associated with the inhibition of tumor metastasis.

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Figure 6.  Effect of heparin-polyethylenimine (HPEI)–plasmid interleukin-15 (pIL15) on cell apoptosis and proliferation in vivo. (A) Induction of apoptosis was indicated by TUNEL assay. The TUNEL-positive cells display dark green nuclei and are observed under a fluorescence microgroup (original magnification, ×200), and the percentage of apoptotic cells was determined as described in the Materials and Methods (original magnification, ×200; *< 0.01). (B) The number of cancer cell nuclei that were strongly PCNA positive was counted as a ratio of immunoreactive positive cells to the total number of cells counted. Dramatic reduction of PCNA expression was noted in the HPEI–pIL15 group compared with the three control groups (*P < 0.05) (original magnification, ×400). NS, normal saline.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

In the present study we provided a novel gene therapy protocol for melanoma and colon carcinoma. The selection of vectors always played an important role in the succession of gene therapy protocol. Also, the HPEI nanogels have shown promising application as a gene delivery system.(6) We constructed a plasmid with high expression efficiency of IL15 and utilized the HPEI–pIL15 complexes to induce antitumor activity against lung metastases of B16-F10 melanoma and CT26 colon carcinoma in a therapeutic setting. We found that the HPEI nanogels could be a novel nonviral gene vector and HPEI nanogels delivering pIL15 had promising application in cancer gene therapy.

Interleukin-15 played multiple roles in peripheral innate and adaptive immune cell function. It is a cytokine with strong antitumoral activity, mediated in a variety of ways. Interleukin-15 could not only activate NK cell proliferation, cytotoxicity, and cytokine production, but also promote the formation of tumor-specific cytotoxic CD8+ T lymphocytes.(14,16,24,25) This multitude of basic studies on the biology of IL15 and the models of human disease point toward a potential clinical benefit of IL15. Interleukin-2 has similar in vitro biological effects and shared receptor components with IL15. Moreover, IL2 has been approved by the Food and Drug Administration since 1992 and is effective for increasing lymphocyte subsets at low doses without significant toxicities.(26) In an early study, Munger et al.(27) found that treatment with IL15 resulted in a higher therapeutic index and was much safer than that of IL2. Recently, to analyze the distribution of IL15, a clinical trial is being on study. Also, six clinical trials of IL15 are now being studied on the treatment of melanoma, AIDS and renal cell cancer using recombinant interleukin-15 protein or plasmid IL15 as an adjuvant.

Although IL15 gene therapy has shown promising application, there are still some tough challenges, such as the lack of an ideal gene expression vector and delivery system. Constructing a plasmid with a high expression of protein was very important for excellent gene therapy protocol. In the present study, to increase the secretion of IL15, we inserted an IL2 signal peptide into the upstream of a mature IL15 gene and constructed a pcDNA3.1–IL15 plasmid. Also, IL15 could highly express in the B16-F10 melanoma cells and CT26 colon carcinoma cells and secrete into the medium after transfection with this plasmid. A good gene transfer carrier was another important factor in successful gene therapy protocol. Several gene delivery methods have been used to express IL15 and achieve some effects in the treatment of cancer.(21,28–30) However, concerns of transfection efficiency and safety are associated with the use of these carriers.

Several cationic polymers have been investigated for use as gene carriers, and among them PEI was gaining attention. Many investigators believe that PEI-based DNA transfer systems are well suited for use in gene therapy for the treatment of cancer.(3,31–34) Although PEI has been proven to be effective in gene delivery due to its condensation of DNA, it is not biodegradable. Moreover, the improvement of transfection efficiency is accompanied by increased cytotoxicity. Some scientists have modified PEI using PEG, Pluronic, Poly Lactic Acid or Polycaprolactone, but there were still some disadvantages, such as long half-life in vivo, poor biocompatibility and rapid degradation.(35–37)

Heparin is a biocompatible, nontoxic material. We chemically conjugated low molecular weight PEI with heparin and expected that the heparin conjugation would affect the character of PEI, specifically its cytotoxicity. The transfection efficiency of HPEI nanogels was comparable to PEI25K. Also, the HPEI nanogels had better blood compatibility and lower cytotoxicity compared with PEI2K and PEI25K. Moreover, the HPEI nanogels were stable in vitro and could be quickly degraded into low molecular weight PEI followed by excretion through urine.(6)

Another advantage of this material is that HPEI nanogels might be a good gene carrier to the lung. We compared the distribution level of the HPEI–pIL15 complexes with that of PEI2K–pIL15 and PEI25K–pIL15 in the lung. The real-time PCR results showed that HPEI exhibited the highest plasmid distribution level in the lung. Our results also showed that intravenous administration of HPEI–pIL15 nanogels was able to stimulate an immune response that inhibits the development of tumors. The mice treated with HPEI–pIL15 had a very low tumor metastasis index compared with all other groups without signs of acute inflammatory responses or other pathergia of the heart, liver, spleen and kidney. The reasons might be that it could be quickly degraded into PEI followed by excretion through urine and the rather low dose of 5 μg per time plasmid we chose.

In the present study, we also observed consistent results that many NK cells were intermingled among the tumor cells. Moreover, the production of TNF-α and IFN-γ in the serum and the cytotoxic activity of spleen cells increased significantly in the HPEI–pIL15 group. Some researchers found that IL15 also had antitumor activity independent of NK and CD8+ T cells.(17,38) Activating the other innate cells, such as macrophages, might boost the antitumor responses. Furthermore, the induction of apoptosis and inhibition of cell proliferation in lung tumor foci in this study could be relevant for all of the NK cells, CD8+ T cells, macrophages and other innate immune cells.

In conclusion, our data showed that intravenous delivery of HPEI–pIL15 nanogels was an efficient way to transfer genetic material to the lung. Also, the administration of HPEI–pIL15 complexes led to the significant inhibition of lung metastasis of malignant melanoma and colon carcinoma with no significant systemic toxicity. Therefore, HPEI nanogels delivered by pIL15 might be a new and interesting cancer gene therapy protocol.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

This work is supported by the National Key Basic Research Program (973 Program) of China (No. 2010CB529900) and the China National Science and Technology Programs of Significant New Drugs to Create (No. 2009ZX09103-714).

Disclosure Statement

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

The authors declare they have no competing interests.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
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