Co-polymeric hydrophilic nanospheres for drug delivery: Release kinetics, and cellular uptake
Anita Kamra Verma*, A Chanchal & A Maitra†
Nano-Biotech Laboratory, Kirori Mal College, University of Delhi, Delhi 110 007, India
†Department of Chemistry, University of Delhi 110 007, India
Nanobiotechnology focuses on the biological effects and applications of nanoparticles that include nano-safety, drug encapsulation and nanotherapeutics. The present study focuses on hydrophilic nanospheres of copolymers N-isopropylacrylamide [NIPAAM] and vinyl pyrrolidone [VP], encapsulating a bioactive derivative of 5-fluorouracil-hexyl- carbamoyl fluorouracil (HCFU). The size of the nanospheres was ~58 nm and the surface charge measured was -15.4 mV. Under optimal conditions, the yield was >80%, and the drugloading was 2%. The entrapment efficiency was
~75%. Wide-angle X-ray diffraction analysis showedthat the entrapped HCFU was present in an amorphous state,which has higher water solubility compared with the crystallinestate. Slow drug release from nanospheres wasobserved in PBS and serum, with 80% released at 37°C after72 h. The HCFU loaded polymeric nanospheres have been found to be stable in whole blood having negligible RBC toxicity. Cytotoxicity in Mia-Paca 3, pancreatic cancer cell line was done in a 24-72 h assay. Dose dependant cytotoxicity was observed when incubated with various concentrations of HCFU loaded polymeric nanospheres while HCFU per se (<1 mg) showed 90% toxicity within 24 h.
Keywords: Cytotoxicity, Co-polymeric nanospheres, Hexyl carbamoyl fluorouracil (HCFU), Mia-Paca 3 human pancreatic cell line.
Nanobiotechnology is a hybrid discipline, a unique amalgamation of biotechnology and nanotechnology that can simulate or incorporate biological systems at the molecular level. Biotechnology uses the knowledge and techniques of biology to manipulate molecular, genetic, and cellular processes to develop products and services, and is used in diverse fields from medicine to agriculture. The nanotechnology market is predicted to be valued at $ 1 trillion by 2012, so the likelihood of exposure to synthesized nanomaterials will exponentially increase. Utility of nanotechnology to biomedical sciences imply creation of materials and devices designed to interact with the body at sub-cellular scales with a high degree of specificity. This could potentially be translated into targeted cellular and tissue-specific clinical applications aimed at maximal therapeutic benefits with very limited adverse-effects. Nanotechnology in biomedical sciences presents many revolutionary opportunities in the fight against all kinds of cancer, cardiac and neurodegenerative disorders, infection and other diseases. Currently the area of
nanomedicine showing the greatest potential is the use of tiny polymeric drug carriers. Nanoparticles of biodegradable polymers are essentially the ideal particulate system to promote an idealistic chemotherapeutic regime having a slow, sustained, controlled and targeted drug delivery system. Micro- or nano-particulate devices composed of hydrogels or containing hydrogel components may have an advantage as they may have improved swelling kinetics involving thermo-sensitivity of drug release property and a wide variety of biomedical applications due to their small size.
The potential use of hydrogel nanospheres and polymeric nanoparticles as carriers for efficient or targeted delivery of drugs has now been successfully established1-3. These particles facilitate the passive targeting of anticancer drugs to the tumor tissue through enhanced permeation and retention (EPR) effect4. The size and surface hydrophilicity are crucial factors in drug targeting5,6. Several efforts have been made to overcome delivery problems, and to reduce the toxic effects of anticancer drugs by chemical modification. This study involves the development of a transient hydrophobic derivative 5-fluorouracil- hexyl-carbamoyl fluorouracil (HCFU), with enhanced antitumour activity, so as to encapsulate it in
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*Correspondent author Telephone: +91-9818921222 Fax: 011-27666579
E-mail: akamra23@hotmail.com; akverma@kmc.du.ac.in
polymeric nanospheres that are known to be water dispersible biocompatible, and non-antigenic.
Pancreatic cancer is the fifth leading cause of cancer-related mortality in the United States, with an estimated 33,370 deaths attributable to this disease in 2007. In India, there were 14,230 cases of pancreatic cancer in the year 2001 which reached 17,000 during 2009.
5-Fluorouracil (5-FU) is known for its broad spectrum antineoplastic activity and has been used in clinics for about 40 years7 against carcinomas of pancreas, breast, head, neck and ovary8-12. However, this antitumor agent exhibits toxicity in bone marrow and gastrointestinal tract12. The palliative chemotherapy by gemcitabine, oxaliplatin and fluorouracil (5-FU) may be used to improve quality of life and gain a modest survival benefit of the patient.
As a pyrimidine analogue, 5-FU is transformed inside the cell into different cytotoxic metabolites which are then incorporated into DNA and RNA, finally inducing cell cycle arrest and apoptosis by inhibiting the cell's ability to synthesize DNA. Interestingly, treatment with antimetabolites are palliative and not curative since biochemical differences between the transformed target cells and normal host cells are insufficient for highly selective kill13. Drug toxicity as well as antitumour effects are highly dose dependent and can be modified by route of administration.
In the present study, 1-hexyl-carbamoyl-5- fluorauracil, a derivative of the anti-pyrimidine 5-FU has been encapsulated in co-polymeric nanospheres, and the physical characterization of the HCFU loaded nanospheres such as size, surface charge, drug loading, release kinetics and drug polymer interactions have been evaluated. Its activity against Mia-Paca 3, human pancreatic cancer cell line, have been investigated for a possible therapeutic potential.
Materials and Methods
Materials—N-Isopropylacrylamide (NIPAAM) was purchased from Acros Organics, Belgium and was re-crystallized from N-hexane and stored at 4ºC. N-N' methylene bis-acrylamide (MBA) was bought from Sigma, USA and was used directly without further purification. Ammonium persulphate (APS) and ferrous ammonium sulfate (FAS) were procured from SRL, India. N-vinylpyrrolidone (VP) was purchased from Fluka, USA. VP was freshly distilled before polymerisation. 5-fluorouracil and hexyl- isocyanate were bought from Acros Organics, Belgium. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
2H-tetrazolium bromide (MTT) was purchased from Sigma, USA.
Synthesis of co-polymeric nanospheres—Free radical polymerization reaction was used to synthesize a co-polymer of water soluble monomers of NIPAAM and VP. Reaction was initiated by adding FAS and was crosslinked with MBA using APS as a catalyst. Detailed method of preparation is as follows: 9:1 molar ratio of recrystallized NIPAAM and freshly distilled VP were dissolved in 100 ml of water. To cross-link the polymer chain, 300 µl of MBA (0.049 g/ml) was added in aqueous solution of monomers. Nitrogen gas was passed through for 30 min to remove the dissolved oxygen. Reaction was initiated by adding 50 µl of FAS (5 mg/ml) and 50 µl of saturated solution of APS. The polymerization was done for 8-10 h at 35ºC in nitrogen atmosphere. Total aqueous solution of polymer was then dialyzed at 4ºC for 5 h using a spectrapore dialysis bag (12 kDa cut off). The dialyzed solution of polymeric nanospheres was lyophilized immediately to obtain a dry powder for subsequent use.
Preparation of HCFU—HCFU was prepared as per Ozake et al14. Briefly, hexyl-isocyanate (19.08g, 0.15 M) and 5-FU (13.0 g, 0.1 M) were heated in 40 ml of pyridine at 90ºC, for an hour (Fig. 1).
Reaction mixture was cooled to room temperature.
About 30 ml of pyridine was evaporated at 50ºC under reduced pressure. Ethyl alcohol was added at 55ºC and the solution kept overnight at 4ºC. A crystalline crop of HCFU was obtained (22.5 g,
~87.4% yield).
Loading of HCFU—Free HCFU was physically entrapped in co-polymeric nanospheres. A known amount of lyophilized powder was re-dispersed in 10ml of water. HCFU was dissolved in a mixture of chloroform and methanol (in the molar ratio 9:1) and was added drop-wise to the aqueous solution with vigorous stirring followed by cold bath sonication to remove the organic solvents. The loading efficiency was calculated in terms of mg of the drug per 100 mg
Fig. 1—Schematic representation for preparation of HCFU.
of polymeric nanospheres. Owing to the hydrophobic nature, HCFU preferentially went into the hydrophobic core of the polymeric nanospheres. The HCFU loaded polymeric nanospheres (HLPN) were then lyophilized to dry powder and stored at 4°C.
Entrapment efficiency (EE)—The HCFU loaded nanoparticles were separated from unentrapped HCFU after passing the solution through a Millipore filter UFP2THK24 [100 kDa cut off] and absorbance of free HCFU was noted using the UV-1601, UV visible spectrophotometer (Bio-Tek) at 262 nm. The EE (%) was calculated as:
total free
total
([HCFU] [HCFU] )
EE (%) 100
[HCFU]
= − ×
where, ([HCFU]total and [HCFU]free) are the amount of total HCFU added and free HCFU respectively.
Characterization of co-polymeric nanospheres
Dynamic light scattering (DLS) and zeta potential—The size measurements (hydrodynamic radius, Rh) were done by dynamic laser light scattering (DLS) having excitation source of He-Ne gas laser with a wavelength of 633 nm and a power of 4mW in linearly polarized single frequency mode, using Zeta Nano-ZS, backscattered (Malvern Instruments, USA). The count rate was ~200 kcps which was adjusted by the instrument itself by changing the attenuator position. The surface charge was also measured.
FT-IR—For the transmission FTIR spectrum, the lyophilized powder of polymer, void nanospheres and HLPN were milled with KBr to form pellets. IR spectra of the pellets were recorded using Perkin Elmer, UK RX1 model (Resolution 4 cm-1; KBr Beam-splitter; Source MIR; Detector LiTaO3).
X-ray diffraction (XRD)—Powder XRD spectra of the samples, both void nanospheres and HLPN were taken on Phillips Xpert PW 183098 Model spectrometer.
Morphological assessment of size by transmission electron microscopy (TEM) and scanning electron microscopy (SEM)—The Electron microscopy of co-polymeric nanospheres was done in a Phillips EM300 instrument using the desired magnification.
The lyophilized powder was redispersed in methanol and mounted on a carbon-coated grid. The grid was dried in a desiccator at room temperature before loading on the microscope.
The external surface of the nanospheres were viewed by Scanning Electron Microscopy (SEM) [Jeol/JSM-35 CF] after a layer of gold was deposited by evaporation (Fine Coat, Ion splutter JFC 1100, Jeol, Paris, France)
Release kinetics—Lyophilized HCFU loaded co-polymeric nanospheres (50 mg) was redispersed in 10 ml buffer at physiological pH and kept at a constant temperature. Every hour 500 µl of the solution was filtered through a Millipore filter of 100 kDa cut off as described earlier. The same methodology was repeated with undiluted serum.
Assessment of biological activity
Haemolytic activity—Haemolytic activity was done on whole blood. Briefly, the heparinized blood procured from normal subjects and red blood cells were washed twice with phosphate buffered saline (PBS, pH 7.4) prior to the assay. Human RBC (100 µl) suspended with 1:1 ratio in PBS were incubated with HCFU, void nanospheres and HLPN upto 4 h at 37ºC. The absorbance of the lysed RBC was read in a UV visible spectrophotometer (Bio-Tek) at 410 nm. Percent haemolysis was calculated at various time points as per established protocols15. Treatment with 0.01% Triton X was considered as maximum.
In vitro cytotoxicity and light microscopy—Mia- Paca 3, human pancreatic cancer cell line (5×103 cells/well) were incubated in 100 µl of RPMI-1640 supplemented with 10% FCS, 2 mM L-glutamine, with various concentration of HCFU, void nanospheres and HCFU loaded nanospheres (HLPN).
Briefly, cells were grown at 37ºC in a humidified atmosphere of 5% CO2. Cytotoxicity was measured by the tetrazolium (MTT) method16. Exponentially growing cells were exposed to various concentrations of HCFU per se, HLPN and void nanospheres. After the treated cells were incubated for 24, 48 and 72 h respectively, 20 µl MTT (5 mg/ml) was added, and the plates were incubated at 37ºC for 4 h. To dissolve formazan, 150 µl DMSO was added, and the plates were measured at 540 nm by a spectrometer. All measurements were done in triplicates. The relative cell viability (%) related to control wells containing cells without nanospheres was calculated as per published protocol17:
Relative cell viability (%) = [A]test/[A]control × 100
where, [A]test is absorbance of the test sample and [A]control is the absorbance of the control sample.
By using the non-radioactive assay for assessing the proliferation of cells we were able to quantify the amount of MTT cleaved, which is directly proportional to the viable cell population.
The plates were viewed under an Olympus inverted Microscope (Model CKX41) and the triplicates photographed before addition of MTT.
Statistical analysis—Data have been represented as mean of 3 sets of experiments, with the standard deviations. Student’s t-test was used to evaluate the differences between experimental groups. P values were taken to be statistically significant.
Results and Discussion
Amphiphilic co-polymeric nanospheres were successfully prepared with a hydrophobic core and a hydrophilic outer shell. Highly effective drugs are characterized by low solubility that hinders their pharmacological efficacy. Table 1 clearly indicates the altered characteristics of HCFU as compared to the original drug 5-fluorouracil. The loading percent achieved was 2% (w/w) with ~75% entrapment efficiency. Co-polymeric nanospheres composed of NIPAAM-VP have been found to have important potential applications for the administration of therapeutic molecules because of their biocompatibility and non-antigenicity, as discussed in earlier studies18.
Monomers of NIPAAM and VP were subjected to radical polymerization, thus forming a chemically crosslinked amphiphilic nanosphere. The hydrophobic core consists of the isopropyl groups of NIPAAM and the long carbon chains of the polymer inside the nanospheres and hydrophilic outer shell was composed of hydrated amides and pyrrolidone groups projected from the monomeric units. This amphiphillic nature of the nanospheres has been exploited to encapsulate the water insoluble derivative HCFU inside the hydrophobic core. Physical combinations of drugs with polymers are normally achieved by encapsulation of drugs in polymeric nanospheres or hydrogels19-21. The yield of the polymeric nanospheres was approximately 80%.
The FTIR characteristic absorption peaks for vinyl bond of monomers appeared at 800-1000 cm-1. Peaks of corresponding stretching mode of vinyl bonds disappeared in the polymer spectrum, indicating that polymerization has taken place. Peaks at 1640- 1720 cm-1 are corresponding to >C = O stretching, and 1640 cm-1(C = C stretching). The IR absorption bands in the region of 1440 cm-1 could possibly be due to the bending vibrations of –CH3 group. Being a hydrogel, the water of hydration attached to the polymer gives rise to a broad and intense peak at 3435 cm-1 (Fig. 2)
Powdered XRD measurements were done to confirm the crystallinity of the void nanospheres and HLPN. The XRD measurements are shown in Fig. 3.
The spectra shows the crystalline nature of the carrier co-polymeric nanospheres, but HCFU remains in a fluid state inside the hydrophobic core. Sharp crystalline peak of HCFU in the range of 2θ = 20°
were absent in the encapsulated co-polymeric nanospheres. The fluid state of HCFU in the nanospheres enables the release of the drug from the encapsulated nanospheres.
HCFU (2% w/w) was entrapped in lyophilized powder of co-polymeric nanospheres dispersed in aqueous buffer (pH 7.4). The absorbance of the entrapped drug HCFU, and HCFU (un-entrapped) was taken at 262 nm and the entrapment efficiency (EE%) was calculated to be 75% (Fig. 4). Entrapment efficiency reduces drastically on increasing the loading percent beyond 2% (w/w). HCFU is an amphiphilic molecule, with 5FU as the hydrophilic moiety, and the hexyl chain being the hydrophobic moiety. Hence, it is expected that perhaps, HCFU remains dissolved at the interface, with 5-FU component projected at the hydrophilic outer shell of the nanospheres. HCFU is sparingly soluble in water indicating that after saturated loading of the drug, i.e.
2% (w/w) in the polymeric nanospheres, most of the HCFU molecules were precipitated.
The small size of 58 nm of the void nanospheres and 62 nm of the HCFU loaded polymeric nanospheres achieved are ideal for drug delivery. The
Table 1—Characteristics of HCFU and 5-FU
Compound Melting Point UV-Spectral Data Solubility Mol. Weight Status
5-fluorouracil 280-284°C λmax 262 Hydrophilic 130.08 Original drug
1-Hexylcarbamoyl FU 105-110°C λmax 258 Hydrophobic 256.9 Altered drug
average size of the HLPN calculated from their diffusion co-efficient using Stoke Einstein’s equation at 25°C, and was around 62 nm (Fig. 5), which is slightly larger than the void nanospheres (58 nm).
Each measurement was repeated 2-3 times for size.
NIPAAM is highly temperature dependent, and its properties can be tailored by adding co-monomers. Its lower critical solubility temperature (LCST) was known to be around 32°C, but in the present case it was seen that beyond 39°C its size increases drastically (unpublished data). TEM and SEM picture (Fig. 6) shows that the particles are more or less spherical in shape. They retain their shape although a bead-necklace type of effect is seen. Also, the
intermolecular electrostatic repulsion forces obviously hinder the inter-molecular co-aggregation.
The surface charge (zeta potential) of the nanosphere was found to be -15.4 mV (Fig. 7). The averaging ζ potential of the polymer molecules has to be approximately between -9 and -16 mV (in 1 mM NaCl) to enable the production of nanoparticles. The in vitro release kinetics in buffer and serum, (Fig. 8a and b) showed a slow release of drug with a gradual peak shift indicating hydrolysis of the HCFU to the original drug (5-FU) itself. Initially, about 60% HCFU was released in buffer, after which HCFU was released slowly for about 24 h. The hydrolysis kinetics of 5-FU derivatives have been
Fig. 2—Mid IR–KBr pellet using Perkin Elmer Fourier transformed IR. Absorption peaks for vinyl bond of monomers appeared at 800-1000 cm-1 and disappeared in the polymer spectrum, indicating that polymerization had taken place
studied in various pH and plasma extensively by Burr and Bundgaard22 who reported strict first order release kinetics. Peak shift suggests the possible hydrolysis of the carbamoyl bond of HCFU releasing pure 5-FU, which is in unison with previously published papers on hydrolysis of carbomyl derivatives of 5-FU23.
Fig. 3—XRD of void and HCFU loaded polymeric nanospheres shows the crystalline nature of the carrier polymeric nanospheres
Fig. 4—The UV-Vis spectra of entrapped & free HCFU.
Entrapment efficiency ~75%
Fig. 5—The DLS size distribution of nanospheres having the size 58±6.45 nm
Fig. 6—Transmission (a) and scanning (b) electron microscopic picture of nanospheres
Carbamoyl derivatives are known to be susceptible to enzymatic hydrolysis in plasma. Release kinetics shows hydrolysis in water/buffer and is pH dependant (unpublished data). The increased lipophilicity of HCFU as compared to 5-FU is accompanied by decreased water solubility. The fall in the concentration of the drug over a period of time can be attributed to settling of the drug due to the decreased water solubility. HCFU has shown both lipophilicity and reduced melting points (Table 1). Burr and Bundgaard22 believed that derivatization of 5-FU may lead to compounds possessing both higher lipophilicity due to a decreased intermolecular hydrogen bonding achieved by blocking the 1-NH group in 5-FU by carbamoylation, which manifests in the decrease in melting points.
In serum also, it shows first order release kinetics.
The release in serum is slow possibly due to enhanced stability of the polymeric nanospheres due to absorption and complement proteins present in the serum. Under physiological conditions enzymatic and chemical degradation of polymeric nanospheres takes place by hydrolysis24.
The percentage of haemolysis was measured at different incubation time intervals using various concentrations of HCFU per se, HLPN and void co- polymeric nanospheres at 37°C. The encapsulation of the HCFU in polymeric nanospheres has significantly reduced the RBC toxicity. While 100% haemolysis was observed after incubation with the HCFU per se in 24 h, only ~30% haemolysis was observed by drug loaded polymeric nanospheres and absolutely no haemolysis was observed even after 24 h of incubation with the void nanospheres. (Fig. 9).
The cytotoxic effects of the void nanospheres, free HCFU, 5-FU per se and the HCFU loaded polymeric nanospheres against Mia-Paca 3, pancreatic cancer cells, were tested in vitro using a standard MTT assay.
HCFU has been reported to be more active than 5-FU, against rapid growing tumors-lung carcinoma and melanoma25. But its efficacy against pancreatic cancer had not been assayed. Dose dependant cytotoxicity was observed after 24 h upto 72 h of incubation of HLPN (Fig. 10) while HCFU per se (<1 mg) showed 93% toxicity within 24 h.
Increased antitumor activity has been observed over a period of 72 h. HCFU is known to be active both in rapid growing tumor systems26 and slow
Fig. 7—Zeta potential of nanospheres [-15.4 mV]
Fig. 8—First order release kinetics of HLPN in phosphate buffer saline [10 mM phosphate with 150 mM NaCl] at pH 7.4 (a) and in serum (b).
Fig. 9—Effect of HCFU per se, Placebo and HLPN on RBC toxicity in a 24 h assay.
growing tumors. HCFU was physically entrapped in the polymeric nanospheres and remained active after encapsulation that has been clearly assessed by the MTT assay.
In the present studies, because of the lipophilicity and small size of the molecule HCFU preferentially goes into the hydrophobic core. When re-dispersed in water/buffer, it was found that HCFU being a small molecule simply diffuses out of these nanospheres.
The nanospheres mimic a hydrogel, as hydrogels tend to ‘swell up’ in water as a function of time. The diffusion coefficient of a drug in a hydrogel, is related to its diffusion coefficient in water, its molecular weight, and the percentage of polymer in the gel27. The cell uptake studies clearly indicate the time dependent aggregation of the swollen polymeric nanospheres around the cancer cells. (Fig. 11).
Interestingly, the nanospheres retain their integrity and do not merge thereby slowly releasing their contents leading to time dependent toxicity.
Conclusions
The present study signifies the importance of improving the drug delivery of 5-FU by not only making a transient derivative which is more potent than the original drug, but by encapsulating it in nanospheres, it is possible to infuse five times more HCFU in the encapsulated form with only 30%
cytotoxicity observed after 72 h of incubation. The slight toxicity observed by void nanospheres was perhaps due to the presence of monomer contaminants. The stability of the nanospheres is evident by the negligible RBC toxicity.
The release of the HCFU from polymeric nanospheres in buffer and serum showed exponential
Fig. 10—Effect of HCFU and 5-FU on Mia-Paca line in a 24 h assay.
Fig. 11—Cellular uptake of nanospheres. Nanospheres around the cell surface.
release. To combat a fast growing cancer, the drug has to be rapidly released at the target site. Rapid release of drugs into plasma, and thus altering their concentration profiles can achieve obvious therapeutical benefits.
The metabolic activity of cells is an appropriate approach for assessing the number of viable cells, since the damaged or dead cells are incapable of exhibiting mitochondrial dehydrogenase activity28. In vitro studies on Mia-Paca 3, pancreatic cells showed that the antitumor activity increased over a period of 72 h thereby showing that the drug is still bioactive, also the release is slow from the polymeric nanospheres. Since, enhanced and sustained cytotoxicity was observed, it appears to be a good candidate for in vivo studies. Also, owing to their size, these nanospheres have relatively enhanced circulation times, as they tend to evade the reticulo- endothelial system (RES). The in vitro studies suggested that these co-polymeric nanospheres could possibly be exploited for efficient drug delivery of biopharmaceuticals in cells as well as for increasing drug delivery across cellular barriers. The in vivo studies are underway.
Acknowledgement
This work was supported by a Department of Biotechnology grant-BT/PR/9895/NNT/28/55/2007, for Nano-Science and Nano-Technology Research Initiative, Government of India. A Chanchal is thankful to DBT for Senior Research Fellowship.
Thanks are due to Dr. Rajni Rani, National Institute of Immunology (NII), New Delhi for TEM facility.
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