21September2019

Nano-Micro Letters

Polymer Nanoparticles Prepared by Supercritical Carbon Dioxide for in Vivo Anti-cancer Drug Delivery

Maofang Hua1, Xiufu Hua2,*

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Nano-Micro Letters, , Volume 6, Issue 1, pp 20-23

Publication Date (Web): December 30, 2013 (Article)

DOI: 10.5101/nml.v6i1.p20-23

*Corresponding author. E-mail: hua_xiufu@163.com

 

Abstract

 


Figure 1 (a) Preparation of drug-loaded nanoparticles by precipitation in SC-CO2; (b) SEM image of BSA-PMMA nanparticles.

A new approach for producing polymer nanoparticles made of bovine serum albumin-poly(methyl methacrylate) conjugate by precipitating in supercritical CO2 is reported. The nanoparticles were loaded with the anti-tumor drug camptothecin. With albumin serving as a nutrient to cells, the drug-encapsulated nanoparticle shows an enhanced ability to kill cancer cells compared to that of the free drug in solution both in vitro and in vivo.


 

Keywords

Protein; Polymer; Nanoparticle; Drug delivery

 

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Introduction

 

Polymeric nanoparticles have much medical promise with a large number of therapeutic nanoparticles presently in clinical trials or approved for clinical use [1-4]. Ge et al. [5] developed a new type of protein-polymer hybrid nanoparticles made of hydrophilic denatured bovine serum albumin (BSA) covalently bonded to hydrophobic poly(methyl methacrylate) (PMMA).

 

Based on their study, here we report a new approach for producing such nanoparticles made of bovine serum albumin-poly(methyl methacrylate) conjugate by precipitating in supercritical CO2. BSA-PMMA nanoparticles can be prepared with hydrophobic drugs being encapsulated by a simple precipitation using supercritical carbon dioxide (SC-CO2) as an anti-solvent. SC-CO2 is also non-toxic, non-flammable, and FDA approved. Such a process can readily be scaled to kilogram quantities, and represents a new approach with great potential to produce dry polymeric nanoparticles for sustained drug delivery [6]. In this work we use the solution enhanced dispersion by supercritical fluids (SEDS) method to produce camptothecin (CPT)-encapsulated BSA-PMMA nanoparticles. The nanoparticle shows an enhanced ability to kill cancer cells compared to that of the free drug in solution both in vitro and in vivo.

 

Experimental

 

The BSA-PMMA conjugate was synthesized by the method described by Ge et al.previously [5]. In a typical experiment, BSA (lyophilized powder, from Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO) at 50oC at a concentration of 2 mg/mL, followed by addition of acrylic acid N-hydroxysuccinimide ester (NAS) (in DMSO at a concentration of 20 mg/mL) with a molar ratio of NAS to BSA of 130:1. After reaction at 25oC for 5 hours, methyl methacrylate (MMA) was added, followed by addition of 2,2’-azobis(2-methylpropionitrile) (AIBN) (8 mM) to initiate the polymerization at 70oC. The conjugate was collected by precipitation in a methanol/ethyl ether (1:8, v/v) mixture. The BSA:PMMA weight ratio in the conjugate was determined as ~4:1 by 1H-NMR. Then, 2 mg/mL of BSA-PMMA conjugate and 0.25 mg/mL of CPT were dissolved in chloroform, followed by subjecting to the SEDS process. Briefly, the solution was injected (1 mL/min) through a nozzle with 250 μm internal diameter into a SC-CO2 (150 g/min) at 40 oC and 100 bar. CPT is used for treating a wide range of tumors [7]. However, it is poorly soluble in water [8]. After preparing the CPT-encapsulated BSA-PMMA nanoparticles by precipitating in SC-CO2 (Fig. 1a), we estimated the yield of our SEDS process to be ~88%. The scanning electron microcopy (SEM) image (Fig. 1b) shows the nanoparticles have an average size around 200~300 nm. The size of CPT-loaded BSA-PMMA nanoparticles dispersed in PBS (pH 7.4) at a concentration of 7 mg/mL was measured to be 310 nm (PDI = 0.121) by dynamic light scattering (mean size: 310 nm, size distribution: ±27 nm).

 

Figure 1 (a) Preparation of drug-loaded nanoparticles by precipitation in SC-CO2; (b) SEM image of BSA-PMMA nanparticles.

Figure 1 (a) Preparation of drug-loaded nanoparticles by precipitation in SC-CO2; (b) SEM image of BSA-PMMA nanparticles.

 

Results and discussion

 

The drug loading was determined as 12.5 wt% in the BSA-PMMA nanoparticles. The drug release profile was studied by dispersing the CPT-loaded nanoparticles in a release medium (PBS, pH 7.4 containing 2% (w/v) Tween 80) [9]. As shown in Fig. 2a, at the drug loading ratio of 12.5 wt%, the encapsulated CPT was released from the nanoparticles over a period of 48 hours with a low initial burst. And the encapsulation ratio was determined to be ~10 wt%. At the early stage of drug release, the diffusion of CPT from nanoparticles probably played an important role and resulted in a burst release. At the late stage, the degradation of BSA-PMMA nanoparticles caused more drug molecules to gradually be released.

 

The CPT-encapsulated BSA-PMMA nanoparticles were analyzed for their ability to retard the proliferation of tumor cells. Human colorectal cancer cells HCT116 were plated in 96-well microplates at 5.0 × 103 cells per well in 190.0 μL of complete medium and allowed to adhere under incubation at 37°C and 5% CO2. Twenty-four hours later, HCT116 cells were exposed to CPT in a 10% v/v DMSO solution containing CPT or CPT-encapsulated nanoparticles with the same drug concentration. The final concentration of DMSO in the cell culture medium was 0.5% (v/v), which had no measurable effect on cell viability. Empty nanoparticles were used as a control. The viability of cell populations was then assessed by the MTT method [10] at 72 h. As shown in Fig. 2b, the dose-dependent cytotoxic effect of the CPT solution was evident cumulating in more than 50% HCT116 survival at 62.5 ng/mL after 72 h. The encapsulation of CPT into the nanoparticles resulted in marked improvements of the anti-tumor activities. After 72 h, less than 10% survival was observed at the CPT concentration above 250 ng/mL. Calculated from the experiment, the IC50 for free CPT was around 87.5 ng/mL, while the IC50 for CPT in nanoparticles was only around 23.5 ng/mL. The empty nanoparticles showed excellent biocompatibility. Around 100% cell survival was observed for cells treated with different concentrations of empty nanoparticles.

 

Figure 2 (a) Sustained release of CPT from nanoparticles; (b) In vitro anti-cancer activities of free CPT and CPT nanoparticles.

Figure 2 (a) Sustained release of CPT from nanoparticles; (b) In vitro anti-cancer activities of free CPT and CPT nanoparticles.

 

We tested the in vivo anti-tumor efficiency of CPT-encapsulated BSA-PMMA nanoparticles by i.v. injection into mice with subcutaneous colon cancer tumors. NOG (NOD/LtSz-scid IL2Rγnull) mice (15-16 weeks old) were injected s.c. with 2 × 107 HCT116 colon cancer cells approximately 10-18 days before dosing. Tumor sizes were measured for the duration of the experiment. Treatment was initialized when the mean tumor size reached approximately 300-500 mm3 (day 1). The animals were sorted into 2 groups, each group having six mice. For group one, as the positive control, free CPT was administered by intraperitoneal injection (i.p. injection). The dose of CPT (9 mg/kg) was based on the literature [11]. CPT is very insoluble in aqueous solution and is acutely lethal when given to mice by i.v. injection at 9 mg/kg (0.18 mg/20 g mouse) due to the particulate matter in the drug suspension. Thus, we followed the previous protocol [11] for the administration of free CPT. In the experiment, the CPT was suspended in a vehicle of 0.5% methylcellulose and 0.1% Tween 80 and administered by i.p. injection as an attempt to maximize its efficiency. Mice were given CPT at 9 mg/kg once daily on day 1 and 7 with a dosing volume of 200 µL. For group two, mice were treated with CPT nanoparticles. The dry powder of CPT nanoparticles was suspended in PBS (pH 7.4) at a concentration of 7 mg/mL with the assistance of probe sonication. The nanoparticles showed excellent dispersability in aqueous solution because of the hydrophilic shell of BSA on the nanoparticles. The treatment was administered intravenously by tail vein injection (i.v. injection) once daily on day 1 and 7 at 9 mg of CPT/kg. The small size and narrow size distribution of the nanoparticles in PBS (pH 7.4) made this formulation very suitable for i.v. injection. As shown in Fig. 3, the tumor volume in CPT nanoparticle-treated mice was significantly smaller than that of mice treated with free CPT. At day 30, the median tumor volume of the CPT nanoparticle-treated mice is 448 mm3, which is almost the same compared with the tumor volume at day 1 (437 mm3). However, for free CPT-treated group, at day 30, the median tumor volume 3515 mm3, which is 7 times increased compared with the tumor volume at day 1. Thus, a significant prohibition of tumor growth was observed when using CPT encapsulated nanoparticles compared with free CPT.

 

Figure 3 In vivo anti-tumor efficiency of free CPT and CPT-encapsulated BSA-PMMA nanoparticles.

Figure 3 In vivo anti-tumor efficiency of free CPT and CPT-encapsulated BSA-PMMA nanoparticles.

PMMA is biocompatible but not biodegradable in vivo. In our study, we choose the PMMA as the hydrophobic core of the BSA-PMMA nanoparitcle is because of its good biocompatibility and the hydrophobicity which could be used to encapsulate hydrophobic drugs. A number of studies have tried to develop PMMA-based nanoparticles for drug delivery [12]. No obvious toxicity was observed by i.v. injection of PMMA nanoparticles in vivo [13]. In our study we utilized the BSA which is biodegradable to copolymerize with PMMA. One BSA molecule was covalently bound with about 49 PMMA chains. Each single PMMA chain attached only has 5-7 of repeating units (MMA) in the BSA-PMMA conjugate we used for in vivo study. After the degradation of BSA in vivo, only oligomers of MMA (5-7 of repeating units with 500-700 Da in molecular weight) would exist. In the in vitro and in vivo study, we observed that empty BSA-PMMA nanoparticles have good biocompatibility and no toxicity to cells or mice.

 

In summary, we have developed a new method to produce protein-polymer hybrid nanoparticles for anti-cancer drug delivery. We have demonstrated that the hybrid nanoparticles prepared by precipitating in SC-CO2 have excellent biocompatibility and efficient cell uptake. The preparation process is simple and easy to scale up. As an example of this type of drug delivery vehicles, compared with free drug formulation, the camptothecin-encapsulated BSA-PMMA nanoparticles shows enhanced anti-tumor activity both in vitro and in animals.

 

Acknowledgements

 

The authors gratefully acknowledge the assistance provided by Post Dr. Junfeng Hui (Department of Chemistry, Tsinghua University) with the TEM images experiments.

 

References

 

[1] L. Zhang, F. Gu, J. Chan, A. Wang, R. S. Langer and O. C. Farokhzad, “Nanoparticles in medicine: therapeutic applications and developments”, Clin. Pharmacol. Ther. 83(5), 761-769 (2008). http://dx.doi.org/10.1038/sj.clpt.6100400

[2] R. Wang, Y. Zhang, D. Lu, J. Ge, Z. Liu and R. N. Zare, “Functional protein–organic/inorganic hybrid nanomaterials”, WIERs: Nanomed. Nanobiotech. 5(4), 320-328 (2013). http://dx.doi.org/10.1002/wnan.1210

[3] J. Ge, E. Neofytou, J. Lei, R. E. Beygui and R. N. Zare, “Protein–polymer hybrid nanoparticles for drug delivery”, Small 8(23), 3573-3578 (2012). http://dx.doi.org/10.1002/smll.201200889

[4] J. Ge, E. Neofytou, T. J. Cahill III, R. E. Beygui and R. N. Zare, “Drug release from electric-field-responsive nanoparticles”, ACS Nano 6(1), 227-233 (2012). http://dx.doi.org/10.1021/nn203430m

[5] J. Ge, J. Lei and R. N. Zare, “Bovine serum albumin-poly(methyl methacrylate) nanoparticles: an example of frustrated phase separation”, Nano Lett. 11(6), 2551-2554 (2011). http://dx.doi.org/10.1021/nl201303q

[6] J. Ge, G. B. Jacobson, T. Lobovkina, K. Holmberg and R. N. Zare, “Sustained release of nucleic acids from polymeric nanoparticles using microemulsion precipitation in supercritical carbon dioxide”, Chem. Comm. 46(47), 9034-9036 (2010). http://dx.doi.org/10.1039/c0cc04258g

[7] B. C. Giovanella, J. S. Stehlin, M. E. Wall, M. C. Wani, A. W. Nicholas, L. F. Liu, R. Silber and M. Potmesil, “DNA topoisomerase I--targeted chemotherapy of human colon cancer in xenografts”, Science 246(4933), 1046-1048 (1989). http://dx.doi.org/10.1126/science.2555920

[8] B. Ertl, P. Platzer, M. Wirth and F. Gabor, “Poly(D,L-lactic-co-glycolic acid) microspheres for sustained delivery and stabilization of camptothecin”, J. Controlled Release 61(3), 305-317 (1999).

http://dx.doi.org/10.1016/S0168-3659(99)00122-4

[9] C. L. Dora, M. Alvarez-Silva, A. G. Trentin, T. J. de Faria, D. Fernandes, R. da Costa, M. Stimamiglio and E. Lemos-Senna, “Evaluation of antimetastatic activity and systemic toxicity of camptothecin-loaded microspheres in mice injected with B16-F10 melanoma cells”, J. Pharm. Pharm. Sci. 9(1), 22-31 (2006). http://www.ualberta.ca/~csps/JPPS9(1)/Senna.E/B16-F10.htm

[10] M. Ferrari, M. C. Fornasiero and A. M. Isetta, “MTT colorimetric assay for testing macrophage cytotoxic activity in vitro”, J. Immunol. Methods 131(2), 165-172 (1990). http://dx.doi.org/10.1016/0022-1759(90)90187-Z

[11] J. Cheng, K. T. Khin and M. E. Davis, “Antitumor activity of beta-cyclodextrin polymer-camptothecin conjugates”, Mol. Pharm. 1(3), 183-193 (2004). http://dx.doi.org/10.1021/mp049966y

[12] A. Bettencourt and A. J. Almeida, “Poly(methyl methacrylate) particulate carriers in drug delivery”, J. Microencapsulation 29(4), 353-367 (2012). http://dx.doi.org/10.3109/02652048.2011.651500

[13] C. Passirani, G. Barratt, J. P. Devissaguet and D. Labarre, “Long-circulating nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate)”, Pharm. Res. 15(7), 1046-1050 (1998). http://dx.doi.org/10.1023/A:1011930127562

 

References

[1] L. Zhang, F. Gu, J. Chan, A. Wang, R. S. Langer and O. C. Farokhzad, “Nanoparticles in medicine: therapeutic applications and developments”, Clin. Pharmacol. Ther. 83(5), 761-769 (2008). http://dx.doi.org/10.1038/sj.clpt.6100400

[2] R. Wang, Y. Zhang, D. Lu, J. Ge, Z. Liu and R. N. Zare, “Functional protein–organic/inorganic hybrid nanomaterials”, WIERs: Nanomed. Nanobiotech. 5(4), 320-328 (2013). http://dx.doi.org/10.1002/wnan.1210

[3] J. Ge, E. Neofytou, J. Lei, R. E. Beygui and R. N. Zare, “Protein–polymer hybrid nanoparticles for drug delivery”, Small 8(23), 3573-3578 (2012). http://dx.doi.org/10.1002/smll.201200889

[4] J. Ge, E. Neofytou, T. J. Cahill III, R. E. Beygui and R. N. Zare, “Drug release from electric-field-responsive nanoparticles”, ACS Nano 6(1), 227-233 (2012). http://dx.doi.org/10.1021/nn203430m

[5] J. Ge, J. Lei and R. N. Zare, “Bovine serum albumin-poly(methyl methacrylate) nanoparticles: an example of frustrated phase separation”, Nano Lett. 11(6), 2551-2554 (2011). http://dx.doi.org/10.1021/nl201303q

[6] J. Ge, G. B. Jacobson, T. Lobovkina, K. Holmberg and R. N. Zare, “Sustained release of nucleic acids from polymeric nanoparticles using microemulsion precipitation in supercritical carbon dioxide”, Chem. Comm. 46(47), 9034-9036 (2010). http://dx.doi.org/10.1039/c0cc04258g

[7] B. C. Giovanella, J. S. Stehlin, M. E. Wall, M. C. Wani, A. W. Nicholas, L. F. Liu, R. Silber and M. Potmesil, “DNA topoisomerase I--targeted chemotherapy of human colon cancer in xenografts”, Science 246(4933), 1046-1048 (1989). http://dx.doi.org/10.1126/science.2555920

[8] B. Ertl, P. Platzer, M. Wirth and F. Gabor, “Poly(D,L-lactic-co-glycolic acid) microspheres for sustained delivery and stabilization of camptothecin”, J. Controlled Release 61(3), 305-317 (1999).

http://dx.doi.org/10.1016/S0168-3659(99)00122-4

[9] C. L. Dora, M. Alvarez-Silva, A. G. Trentin, T. J. de Faria, D. Fernandes, R. da Costa, M. Stimamiglio and E. Lemos-Senna, “Evaluation of antimetastatic activity and systemic toxicity of camptothecin-loaded microspheres in mice injected with B16-F10 melanoma cells”, J. Pharm. Pharm. Sci. 9(1), 22-31 (2006). http://www.ualberta.ca/~csps/JPPS9(1)/Senna.E/B16-F10.htm

[10] M. Ferrari, M. C. Fornasiero and A. M. Isetta, “MTT colorimetric assay for testing macrophage cytotoxic activity in vitro”, J. Immunol. Methods 131(2), 165-172 (1990). http://dx.doi.org/10.1016/0022-1759(90)90187-Z

[11] J. Cheng, K. T. Khin and M. E. Davis, “Antitumor activity of beta-cyclodextrin polymer-camptothecin conjugates”, Mol. Pharm. 1(3), 183-193 (2004). http://dx.doi.org/10.1021/mp049966y

[12] A. Bettencourt and A. J. Almeida, “Poly(methyl methacrylate) particulate carriers in drug delivery”, J. Microencapsulation 29(4), 353-367 (2012). http://dx.doi.org/10.3109/02652048.2011.651500

[13] C. Passirani, G. Barratt, J. P. Devissaguet and D. Labarre, “Long-circulating nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate)”, Pharm. Res. 15(7), 1046-1050 (1998). http://dx.doi.org/10.1023/A:1011930127562

 

Citation Information

Maofang Hua and Xiufu Hua, Polymer Nanoparticles Prepared by Supercritical Carbon Dioxide for in Vivo Anti-cancer Drug Delivery. Nano-Micro Lett. 6(1), 20-23 (2014). http://dx.doi.org/10.5101/nml.v6i1.p20-23

History

Received 19 October; accepted 08 November 2013; published online 30 December 2013

 


Additional Info

  • Type of Publishing: JOUR - Journal
  • Title:

    Polymer Nanoparticles Prepared by Supercritical Carbon Dioxide for in Vivo Anti-cancer Drug Delivery

  • Author: Maofang Hua, Xiufu Hua
  • Year: 2014
  • Volume: 6
  • Issue: 1
  • Journal Name: Nano-Micro Letters
  • Publisher: OPEN ACCESS HOUSE SCIENCE & TECHNOLOGY
  • ISSN: 2150-5551
  • URL: http://www.nmletters.org/current-issue/item/308-polymer-nanoparticles-prepared-by-supercritical-carbon-dioxide-for-in-vivo-anti-cancer-drug-delivery
  • Abstract:

    A new approach for producing polymer nanoparticles made of bovine serum albumin-poly(methyl methacrylate) conjugate by precipitating in supercritical CO2 is reported. The nanoparticles were loaded with the anti-tumor drug camptothecin. With albumin serving as a nutrient to cells, the drug-encapsulated nanoparticle shows an enhanced ability to kill cancer cells compared to that of the free drug in solution both in vitro and in vivo.

  • Publish Date: Monday, 30 December 2013
  • Start Page: 20
  • Endpage: 23
  • DOI: 10.5101/ nml.v6i1.p20-23