Targeted and intracellular delivery of paclitaxel using multi-functional polymeric micelles

ARTICLE IN PRESS

Biomaterials 28 (2007) 1730–1740 www.elsevier.com/locate/biomaterials

Targeted and intracellular delivery of paclitaxel using multi-functional polymeric micelles Wei Yang Seowa, Jun Min Xuea, Yi-Yan Yangb, a b

Department of Materials Science and Engineering, National University of Singapore, Singapore 117576, Singapore Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, #04-01, Singapore 138669, Singapore Received 26 September 2006; accepted 29 November 2006 Available online 19 December 2006

Abstract Natural paclitaxel (Taxols) is an effective anti-cancer drug, although a critical disadvantage is its non-targeting nature. To address this issue, cholesterol-grafted poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-co-undecenoic acid) was synthesized with different starting monomer ratios via a free radical copolymerization route. Folate was subsequently attached to the hydrophilic segment of the polymer in order to target folate receptors-overexpressing cancer cells. The success of synthesis was confirmed with 1 H-NMR carried out in CDCl3/D2O. Using a membrane dialysis method, the polymer was then self-assembled into micelles whose hydrophobic cores could be utilized to encapsulate paclitaxel, an extremely hydrophobic compound. The polymer had a low CMC of 20 mg/L in water. Dynamic light scattering further showed that the sizes of blank micelles formed from the polymer were below 180 nm at different pH values tested and 220 nm upon drug incorporation. More importantly, it was demonstrated that the micelles exhibited a useful pH-induced thermo-sensitivity, such that drug was released more rapidly at pH 5.0 (acidic endosomal/lysosomal environment) than at pH 7.4 (normal extracellular pH). In vitro cytotoxicity assays performed against KB cells then provided concluding evidences that the cellular uptake of micelles surface-functionalised with folate was indeed enhanced due to a receptor-assisted endocytosis process. This novel polymeric design thus has the potential to be a useful paclitaxel vehicle for the treatment of folate-receptor positive cancers. r 2006 Elsevier Ltd. All rights reserved. Keywords: Paclitaxel; Targeted and intracellular delivery; Micelles; pH- and temperature-sensitive; Folate

1. Introduction Paclitaxel can be extracted from the bark of the Pacific Yew tree (Taxus brevifolia) and is a natural taxane effective against carcinomas of the breast, ovary and lung [1]. However, owing to the cyclic components within its chemical structure, paclitaxel is only sparingly soluble (0.3 mg/L at 37 1C) in aqueous media [2]. This drastically limits its bioavailability and is a widely recognized barrier to the application of natural paclitaxel. A partial solution to its poor solubility lies in an organic formulation (Cremophor EL and dehydrated ethanol) commercially available as Taxols. Unfortunately, the organic medium used has been reported to be able to leach a suspected carcinogen—di-(2-ethylhexyl) phthaCorresponding author. Tel.: +65 68247106; fax: +65 64789084.

E-mail address: [email protected] (Y.-Y. Yang). 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.11.039

late—from the infusion bags frequently used by hospitals [3]. More critically, Cremophor EL is known to induce lifethreatening side effects in up to 30% of the treated patients [4]. The root cause of side effects is clearly due to the nonspecific nature of the present formulation whereby the drug distributes randomly during systemic circulation and effects cells (healthy or cancerous) indiscriminately. Hence, there has been a global quest to develop an efficient vehicle that can deliver paclitaxel exquisitely to the intended site without provoking any adverse reactions. Amphiphilic polymeric micelle is a popular candidate as such a carrier. Huh et al. [5], for example, recently reported that hydrotropic micelles made out of poly(2-(4-vinylbenzyloxy)-N,N-diethylnicotinamide) (PDENA) and PEG could be used to encapsulate paclitaxel. In another study, Suh et al. [6] loaded paclitaxel into PEO–PLGA nanospheres and used this formulation successfully to release the drug in a sustained fashion, so as to arrest the

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proliferation of vascular smooth muscle cells. However, these designs did not provide the possibility for drug targeting. In response, we proposed a novel amphiphilic copolymer—poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-co-undecenoic acid) [P(NIPAAm-coDMAAm-co-UA)]—which could be self-assembled into micelles for the targeted delivery of doxorubicin, a watersoluble anticancer drug [7]. A hydrophobic drug like paclitaxel, however, was unable to be encapsulated into the micelles because of the pale hydrophobicity of their cores. This study is, therefore, motivated where we further conjugate cholesterol to a carboxylic group in the hydrophobic segment of the polymer, followed by folate being attached onto an amine group within the hydrophilic chain segment. The presence of cholesterol thermodynamically drives the aquatic self-assembly of core-shell-structured micelles, which in turn expose a shell decorated with folate molecules for active tumor targeting. The hydrophobic core enhanced by cholesterol can then be utilized to incorporate paclitaxel. Folate was chosen to be the targeting moiety since folate receptors are vastly overexpressed in carcinomas of the ovary, lung and breast, but are highly suppressed in normal tissues [8]. Other advantages of using folate include its affordability and it being nonimmunogenic. To trigger drug release after internalization, the micelles were designed to have a lower critical solution temperature (LCST) that can respond to the pH of the external environment in an adjustable fashion. More specifically, in environments that are characteristically more acidic (e.g. acidic tumor tissues, or within the endosomes/lysosomes [9]), the LCST of the micelles dropped below 37 1C, resulting in both the disruption of the core-shell architecture and the release of drug. Paclitaxel was encapsulated into the micelles, and the therapeutic potential of these micelles were assessed via a series of in vitro cytotoxicity assays against the folate receptors-overexpressing KB cell line. It is anticipated that this synergistic combination of passive (due to ‘‘enhanced permeability and retention’’, or EPR effect [10]) and active (folate receptors-mediated endocytosis) targeting will provide a promising platform for future in vivo applications. 2. Materials and methods 2.1. Materials N-Isopropylacrylamide (NIPAAm, 97%), N,N-dimethylacrylamide (DMAAm, 99%) and 10-undecenoic acid (UA, 98%) were purchased from Sigma-Aldrich. NIPAAm was further purified by re-crystallization (thrice) from n-hexane, while DMAAm and UA were purified by reduced pressure distillation before use. 2-Aminoethanethiol hydrochloride (AET, 98%), N-hydroxysuccinimide (NHS, 97%), folic acid dihydrate (97%) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT, 98%) were all purchased from Sigma-Aldrich and used as received. Other reagents include ammonium persulfate (APS, 99%) from Bio-Rad, cholesterol from TCI-EP Tokyo Kasei, N,N0 -dicyclohexylcarbodiimide (DCC, 99%) from Acros Organics, paclitaxel from LC Labs, RPMI 1640 growth medium (without folic acid) from Invitrogen and KB cells from

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ATCC. All other chemicals and solvents were of analytical grade and were used as received.

2.2. Polymer synthesis Polymer synthesis was built upon methods reported earlier [7], but with several important modifications. The overall scheme is presented in Fig. 1. For clarity, the entire synthesis process will be decomposed into three steps—step I, the synthesis of the bare backbone polymer, P(NIPAAm-coDMAAm-co-UA); step II, the grafting of cholesterol, and step III, the conjugation of folate. The polymers were synthesized with three compositions (polymer a–c), as given in Table 1. Henceforth, the notation used will be such that, any polymeric product after step I synthesis will be denoted by their composition (e.g. polymer a). A ‘C’ will be added on as a suffix after step II synthesis (e.g. polymer a-C), while ‘CF’ will be tagged on after the final step of synthesis (e.g. polymer a-CF). 2.2.1. Synthesis of P(NIPAAm-co-DMAAm-co-UA) (Step I) Polymers a–c were prepared via a free radical copolymerization method, employing APS as the initiator and AET as the chain transfer agent (Fig. 1a). For polymer a, briefly, 8.50 g (75 mmol) of NIPAAm and 2.47 g (25 mmol) of DMAAm were first dissolved in 20 mL of ultrapure water. 1.84 g (10 mmol) of UA was converted to its sodium salt with 10 mL of NaOH (1 M). The above solutions were then combined into a round bottom reaction flask and the pH of the mixture was adjusted to be slightly below 6.8 with 1 M HCl. To degas, the mixture was purged with argon for at least 30 min and sealed. Meanwhile, 0.508 g (2.22 mmol) of APS and 0.050 g (0.44 mmol) of AET were separately dissolved in 10 mL of ultra pure water. APS was then added dropwise to the monomeric mixture under vigorous stirring, followed by AET. Ice was used to cool the exothermic reaction mixture to avoid precipitation. Polymerization was then allowed to occur at 27 1C. After 48 h, the polymeric mixture was diluted with 200 mL of 0.5 M NaOH and purified by dialysis against a large external sink of 0.05 M NaOH using membrane (Spectra/Pors Labs Inc.) with a molecular weight cut-off (MWCO) of 2000 Da. The external sink was replaced twice daily and stirred continuously. After 4 days, the external medium was transitioned to de-ionised (DI) water and left for dialysis for a further 3 days. The DI water was also replaced twice daily. The crude product was harvested by removing water using a rotary evaporator. It was then further dissolved in tetrahydrofuran (THF) and re-precipitated dropwise into a large excess of cold diethyl-ether. The mixture was vigorously stirred for a few hours. The final product was obtained after filtration and vacuum drying at 50 1C to remove traces of solvent. Polymers b and c were prepared similarly. 2.2.2. Grafting of cholesterol onto P(NIPAAm-co-DMAAm-co-UA) (Step II) The weight average molecular weight (Mw) of polymer a was determined by gel permeation chromatography (GPC) using a Waters 2695D separations module fitted with a Waters 2414 refractive index detector. The columns used were a Waters Styragels HR 5E THF 7.8  300 mm column, followed by a Styragels HR1 THF 7.8  300 mm column. THF was used as the mobile phase with an elution rate of 1 mL/ min at 28 1C. As shown in Fig. 1b, Polymer a, NHS, DCC and cholesterol were mixed in the following molar ratio 1:1.5:1.2:2.5. Briefly, polymer a was dissolved in dichloromethane (DCM) and then bubbled with argon for 30 min. Meanwhile, NHS and DCC were separately dissolved in DCM. DCC was first added dropwise to the polymer solution, followed by NHS. Activation was allowed to occur in an inert atmosphere with stirring at room temperature (22 1C). After 24 h, the mixture was filtered to remove the byproduct diclohexylurea. Cholesterol was then dissolved in DCM and added dropwise to the activated polymer solution. Cholesterol grafting was allowed to take place for 48 h in an inert atmosphere with stirring. The reaction mixture was dialysed against DCM using membrane with MWCO of 2000 Da to remove any unconjugated free cholesterol and impurities. The external DCM was replaced daily for 5 days. Polymer

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a

NIPAAm H2C

DMAAm

CH C

m

O

NH

+

CH H3C

UA

C n

O

+

N H3C

CH

H2C

CH

H2C

(CH2)6

o

CH2

CH3

CH2

CH3

AET.HCl (HS-CH2CH2NH2.HCl)

COONa

H2C

C

H2C

DCC/NHS, Cholesterol

H 3C

CH m

H2C

H2C

DCC/NHS, Folate

H2C

O

CH o

CH2

S

H2C

CH

H3C

CH2

C

n O

CH3

CH o

S

CH3

CH3 CH3

H H

H

CH2

CH2

R = Cholesterol or H H N

FOL

(CH2)6

O

CH2

N H3C

H2C

NH2

H3C

CH2 CH3

CH2 CH3

(CH2)6

COOR

O

CH H3C

CH n

CH3

NH

DMSO, 22 ºC, 48 hrs

NH2

CH2

N

C

CH2

(CH2)6

CH3

H3C

CH m

CH2

S

CH2

C

NH

H3C

CH o

COONa

O

CH

c

O

CH3

C

DCM, 22 ºC, 48 hrs

H2C

n

N

CH H3C

CH C

O

NH

APS H2O, 27 ºC, 48 hrs

b

H 2C

CH m

CH2 COOR

HO

O HN

O NH

O

H N

N

NH2 N

N

Folate (FOL)

O

Fig. 1. Synthetic scheme of folate-conjugated P(NIPAAm-co-DMAAm-co-UA)-g-cholesterol. (a) Synthesis of P(NIPAAm-co-DMAAm-co-UA) backbone, chain terminated by AET. (a and b) Grafting of cholesterol, (b and c) conjugation of folate.

Table 1 Compositions of polymers Polymer

Feed ratio of NIPAAm:DMAAm:UAa

Actual ratio of NIPAAm:DMAAmb

Actual number of cholesterol molecule/chainb

a-CF b-CF c

3.75:1.25:0.5 3.60:1.40:0.5 3.50:1.50:0.5

2.89b (3.00)a 2.45 (2.57) 2.23 (2.33)

0.92 (1.00) 0.95 (1.00) —

a

Theoretical feed molar ratio. Actual ratio obtained based on relative peak areas of the respective 1H NMR plots. APS and AET were used at 2 and 0.4 mol% of monomer feed.

b

a-C was harvested by evaporating DCM at room temperature. Polymer b-C was also prepared using a similar protocol. 2.2.3. Folate conjugation to P(NIPAAm-co-DMAAm-co-UA)-gcholesterol (Step III) Polymer a-C, folate, NHS and DCC were mixed in the following molar ratio 1:15:30:18 (Fig. 1c). Folate was first activated in anhydrous DMSO using NHS/DCC chemistry for 24 h in an inert atmosphere at 22 1C.

Meanwhile, polymer a-C was dissolved in a mixed solvent comprising DMSO and THF. After filtration, the activated folate was added dropwise to the above polymer a-C solution. Conjugation was allowed to take place for 48 h in an inert atmosphere with stirring. Dialysis was then done against DMSO with daily replacement using membrane with MWCO of 2000 Da. After 5 days, the external medium was transitioned to DCM to exchange for DMSO within the bag for easy evaporation later. Polymer aCF was harvested after 3 days. Polymer b-CF was synthesized similarly.

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2.3. Polymer characterization 2.3.1. Chemical structure—nuclear magnetic resonance (NMR) All samples were prepared by dissolving about 5 mg of polymer in 0.5 mL of a chosen solvent (i.e. CDCl3 or D2O). The 1H spectrum of each sample was recorded with a Bruker 400 MHz Ultrashield Plus and analyzed with in-house software. Chemical shifts were expressed as parts per million, ppm (d). 2.3.2. pH sensitive LCST—UV transmittance Polymeric micelles were made at different pH by a solvent exchange dialysis method. Briefly, 15 mg of polymer were dissolved in 3 mL of DMAc and dialysed (MWCO 2000 Da) against 600 mL of a chosen pH buffer solution (at pH 5.0, 6.0 or 7.4) for 4 days, with daily replacement. Phosphate-buffered saline (PBS) was used to maintain pH at 7.4 and 6.0, while neutralized phthalate buffer was used for pH 5.0. All buffers were freshly prepared with a final ionic strength of 154 mM before use. A UV-Vis spectrometer (Jasco V-570) fitted with a temperature controller (Jasco PSC-498 T) was used to monitor the percentage transmittance of light at 500 nm through the polymeric solution, as a function of temperature (70.1 1C). The LCST of the micelles at that particular pH was then given by the temperature at which the micellar solution displayed 50% transmittance. The heating rate used was 1.0 1C/min and all samples were allowed to equilibrate to the set temperature prior to transmittance measurements. Buffers of different ionic strength (0.154 and 0.167 M) were also used to study for its effect on the LCST. 2.3.3. Particle size—dynamic light scattering (DLS) The hydrodynamic particle size of the micelles in buffer solution was measured at 25 1C by DLS using a ZetaPALSs particle sizing analyzer (Brookhaven Instruments) equipped with a He–Ne laser beam at 658 nm (scattering angle: 901). All samples were filtered with 0.45 mm syringe filters prior to measurements. Each measurement was also repeated 10 times and an average value reported.

were then drawn, one to the curve at high concentrations and another through the points at low concentrations. The CMC value was taken from the intersection between the two tangents.

2.4. Drug loading and in vitro release 2.4.1. Preparation of paclitaxel-loaded micelles Paclitaxel was loaded into the cores of the micelles via a membrane dialysis technique. Briefly, 10 mg of polymer was mixed with 1 mg of paclitaxel in 5 mL of DMF. The resultant mixture was dialysed (MWCO ¼ 3500 Da) against 800 mL of DI water maintained in a 4 1C water bath. The external DI water was replaced daily for 3 days, after which the drug-loaded micelles were harvested by freeze drying. 2.4.2. Determination of drug loading level Two milligram of the drug-loaded micelles was first dissolved in 1 mL of chloroform. Two milliliter of diethyl ether was then added to the above solution to precipitate polymer. The resultant mixture was centrifuged at 10,000 rpm for 30 min, and the supernatant was removed and air-dried. Two milliliter of methanol was added to dissolve the dried sample, filtered with a 0.2 mm syringe filter and analyzed for its paclitaxel concentration using high-performance liquid chromatography (HPLC). The HPLC system consisted of a Waters 2690 separation module fitted with a Waters 996 Photodiode Array Detector and Waters SymmetryShieldTM RP8, 4.6  150 mm column. The mobile phase used was a mixture of ammonium acetate solution (20 mM), methanol and acetonitrile in the volume ratio of 35:20:45. The elution rate was 1.0 mL/min and the paclitaxel detection wavelength was set at 229 nm. The column and sample temperature was maintained at 28 and 20 1C, respectively. A calibration line was constructed to determine paclitaxel concentration in the range of 5–50 mg/L, and the r2-value of peak area intensity plotted linearly against paclitaxel concentration was at least 0.99. The drug-loading level of the micelles was then calculated based on the following formula: Loading level ðwt%Þ ¼

2.3.4. Micellar morphology—scanning electron microscopy (SEM) A drop of drug-loaded micelles dissolved in DI water was dripped onto the surface of a freshly prepared Si wafer and air dried at room temperature for 4 h. The wafer was then mounted onto an Al sample holder and sputter-coated with Pt. SEM observations were carried out with a Jeol JSM-7400F microscope equipped with a field emission gun, at a magnification of 65,000  and with an electron energy of 10 keV. 2.3.5. Tg and decomposition profile—thermal analysis (DSC and TGA) The glass transition temperature (Tg) of polymer a-CF was estimated by differential scanning calorimetry (DSC). A DSC Q100 (Research Instruments Pvt. Ltd.) was used and a heat/cool/heat cycle was employed, at 3 1C/min between 30 and 200 1C. Thermo-gravimetric analysis (TGA) of polymer a-CF was also performed by using a Pyris 1 thermogravimetric analyzer (Perkin Elmer) to obtain its decomposition profile as a function of temperature. The heating schedule was from 35 to 600 1C, at a rate of 2 1C/min in flowing air (30 mL/min). 2.3.6. Critical micelle concentration (CMC) The critical micelle concentration (CMC) of polymer a-CF in DI water was estimated by fluorescence spectroscopy using pyrene as a hydrophobic fluorescence probe. Twenty-five samples of polymer solution with concentrations ranging from 1  105 to 1 g/L were prepared and then left to equilibrate with a constant pyrene concentration of 6.16  107 M for 48 h at 22 1C. Fluorescence spectra of pyrene were recorded with a Fluorologs LFI 3751 fluorescence spectrometer (Jobin Yvon Horiba) at room temperature. The excitation wavelength used was 340 nm and the emission spectra were recorded from 360 to 450 nm. Both excitation and emission bandwidths were set at 2 nm. The peak height intensity ratio (I3/I1) of the third peak (I3 at 393 nm) to the first peak (I1 at 373 nm) was plotted against the logarithm of polymer concentration. Two tangents

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mass of paclitaxel extracted from micelle  100%. mass of drug loaded micelle initially used

2.4.3. In vitro drug release profile In vitro release of paclitaxel from the micelles was studied at different pH as a function of time. Two milligram of drug-loaded micelles was dispersed in 2.5 mL of buffers at pH 7.4 or 5.0. The solution was then filled into a dialysis tubing with MWCO of 8000 Da and submerged fully into a beaker containing 60 mL of the corresponding buffer solution. The beaker was maintained in a 37.070.1 1C water bath and stirred at 100 rpm. At regular time intervals, the entire external medium was removed for HPLC analysis of its paclitaxel content and replaced with fresh buffer. Paclitaxel was extracted from the removed medium after partitioning into an organic phase. Six milliliter of DCM was added to the medium, vortexed vigorously for 5 min and left to stand for 15 min. Upon phase separation, the denser organic DCM layer was carefully drip-separated from the aqueous buffer phase and allowed to evaporate at room temperature overnight. The dried sample containing paclitaxel was then dissolved in 2 mL of methanol and analyzed by HPLC. A profile showing the cumulative amount of drug release as a function of time was plotted at each pH. The experiment was repeated in triplicate. To determine the paclitaxel extraction efficiency of the method described, a standard containing a known concentration of paclitaxel was prepared. Identical methods of extraction were followed and the extraction efficiency was determined to be 90% after HPLC analysis.

2.5. In vitro cytotoxicity assay A folate receptor overexpressing carcinoma cell line of the oral cavity (KB cells) was used. All growth media were prepared by supplementing RPMI 1640 (without folic acid) with 5% penicillin-streptomycin, 10%

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fetal bovine serum, and sterilized with 0.2 mm filter prior to use. The density of the cell line solution was first measured using a haemocytometer and then seeded into 96 well plates at 104 cells per well. The plates were incubated in a humidified 37 1C environment with 5% CO2 for 24 h. Meanwhile, samples of different drug formulation were dissolved in growth medium and then serially diluted to give a range of final drug concentration from 0.01 to 0.5 mg/L. At a suitable time, the cells were exposed to either 0.1 mL of the respective drug samples, or varying concentrations (0–20 mg/L) of polymer without any drug loaded. They were then incubated for another 48 h before replacing the medium within each well with 0.1 mL of fresh growth medium and 20 mL of MTT solution. After incubation for another 3 h, 0.15 mL of DMSO was added to each well to dissolve any formazan crystals formed. The plates were vigorously shaken before measuring the relative color intensity using a microplate reader (PowerWave X, Bio-Teks Inc.). A test wavelength of 550 nm and a reference wavelength of 690 nm were used. The intensity of each well was then given by: (absorbance550 nm–absorbance690 nm). Upon taking the average of each column, cell viability for that particular concentration of sample was expressed as a percentage of the intensity of the controls7standard deviation. Each experiment was repeated 8 times at each polymer concentration.

2.6. Folate competition assays KB cells were exposed to a constant drug concentration of 0.15 mg/L, while being maintained in growth media supplemented with free folate molecules within the concentration range of 0–500 mg/L. The viabilities of the cells were then quantified, as described above. Each experiment was also repeated 8 times at each polymer concentration.

3. Results and discussion 3.1. Polymer synthesis and characterization 3.1.1. 1H NMR analysis of P(NIPAAm-co-DMAAm-coUA) Upon comparing with the 1H NMR spectra of the individual monomers (not shown), one of the first observation in the spectrum of polymer a (Fig. 2) is the absence of the (H2CQCH) vinylic proton signals at around d 5.4–6.6. This is an indication of the success of copolymerization. Based on further comparisons, signals a

and b have been assigned to the iso-propyl group of NIPAAm [CH(CH3)2 at d 4.01 and CH(CH3)2 at d 1.15, respectively], while signal c (d 2.92) has been assigned to the –N(CH3)2 group of DMAAm. This reflects the similarities in the chemical environments of these protons. In addition, it should be noticed that proton d is chemically similar to d0 , while e is comparable to e0 . With this observation, the broad peak at d 1.45–1.95 (signals d+d0 ) has been assigned to –CH2–CH–, while d 2.51 (signals e+e0 ) has been assigned to –CH2–CH– due to their closer proximity to an electronegative CQO group. The characteristic peaks of UA do not appear in the spectrum of the polymer in CDCl3. Instead, signal f has been assigned to the –(CH2)6 group of UA after comparing with the 1H spectrum of pure UA in D2O. The fact that signals due to UA can still be observed in D2O is intriguing. This is because the polymer is expected to self assemble into core-shell nanoparticles in an aqueous D2O environment and UA, being the hydrophobic group, should form the core. It is commonly reported that chemical groups localized within the solid state core can only experience limited molecular motion and consequently do not register NMR signals in D2O [11,12]. In our case, therefore, the observation of UA peaks most probably indicates of a loosely packed core-shell structure, whereby the hydrophobic UA groups can still assess the external environment. In retrospect, but perhaps it should have been with foresight, this suggestion makes good sense since the ability of UA to probe and respond to the pH of the external environment is a pre-requisite for the micelles to exhibit pH sensitivity in the first place. Indeed, this pH responsiveness would be totally lost should UA be strictly confined within the core of the micelles. Nonetheless, we still do not expect all the UA groups to contribute equally to the signal, since some will inevitably be more restricted within the core. Based on integration of the peak areas of signal b relative to signal c, the actual ratio of NIPAAm to

Fig. 2. 1H NMR spectrum of polymer a in CDCl3. d 0 ppm ¼ tetramethyl silane ((CH3)4Si) used as a standard reference; d 7.24 ppm ¼ CHCl3 (atmospheric hydrolysis of CDCl3). Inset is 1H NMR spectrum of polymer a recorded in D2O to show a peak due to UA.

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DMAAm incorporated into the chain can be directly calculated. Table 1 summarises similar calculations done for polymers a–c, and shows that the actual ratio of NIPAAm:DMAAm incorporated is in excellent agreement with the starting feed ratio.

3.1.2. 1H NMR analysis of P(NIPAAm-co-DMAAm-coUA)-g-cholesterol The challenge in this conjugation is to chemically attach only one cholesterol molecule per chain. This was so as not to make the micelles overly hydrophobic and consume all the carboxylic acid groups, which may then interfere with its drug release property and pH-sensitivity later. Therefore, careful control of the feed molar ratio of polymer:DCC:NHS was necessary. As shown in Fig. 3, the general spectrum contour of polymer a-C is very similar to that of polymer a, except for the appearance of several peaks at d 0.65–1. Upon comparison with pure cholesterol in CDCl3 and referencing with standard literature values [13], all the extra peaks can be identified as due to cholesterol and labeled as signals g, h, i and j. This confirms the successful grafting of cholesterol to the polymeric backbone chain. Note also that the methyl (CH3) groups corresponding to signals i and h have been spilt into 2 peaks by the single proton on their adjacent C atom. These signals thus show up as doublets in the spectrum. Signals g and j, however, remain as clear singlets due to the lack of protons in their immediate chemical vicinity. From Table 1, it can be seen that the actual number of cholesterol grafted per chain is close to what is intended. Another noteworthy point is that cholesterol signals are absent in the 1H spectrum performed in D2O (data not shown). This suggests that cholesterol is well isolated within the core, where its molecular motion is further restricted by its rigid and bulky conformation.

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3.1.3. 1H NMR of folate-conjugated P(NIPAAm-coDMAAm-co-UA)-g-cholesterol Fig. 4 shows the proton spectrum obtained in D2O for polymer a-CF and indicates the successful conjugation of folate. Signals k, l, m and n have been assigned to the folate molecule, with signals k and l (at a higher d) being assigned to the aromatic protons that are present in closer proximity to the electronegative N atom. Note also that protons k and l (and m and n) have each been split into 2 peaks by the adjacent proton and thus exist as quartets. Structurally, the observation of peaks due to folate in D2O provides evidence that folate molecules decorate the outer micellar shell and present themselves for targeting purposes. The 1H NMR spectra of polymers b, b-C, b-CF and c are, in general, similar to their polymer a counterparts. 3.2. Molecular weight and thermal properties The polymer used for this study is probably nonbiodegradable. As such, the fate of the polymer after it has released its drug content can be a concern. It has been suggested that the upper limit for the successful filtration through kidney cells is about 40 kDa [14]. This is safely above the weight average molecular weight (Mw) measured for polymer a-CF (19.6 kDa, polydispersity index: 1.78), relative to polystyrene standards. This suggests that the kidneys may be able to filter the polymer from the bloodstream for subsequent excretion. The glass transition temperature of the polymer has been measured to be about 134 1C. A high decomposition onset temperature (300 1C) is also estimated by TGA and suggests that the polymer can be autoclaved prior to in vivo administration. 3.3. Critical micelle concentration The CMC is an effective parameter of micellar stability and a low critical value is desired. In this study, micelle

Fig. 3. 1H NMR spectrum of polymer a-C in CDCl3. Peak at about d 5.3 ppm is most probably due to DCM (CH2Cl2), a solvent used during polymer synthesis. Inset is a blown up of the main spectrum from d 0.65–1.0 to show cholesterol peaks.

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formation was monitored by using pyrene as a hydrophobic probe. Fig. 5a shows several fluorescence patterns obtained from pyrene equilibrated with aqueous solutions of polymer a-CF at various concentrations. Of special interest are the third (I3 at 393 nm) and first (I1 at 373 nm) peaks, since their peak height-intensity ratio (I3/I1) can be used as a sensitive parameter to represent the polarity of the microenvironment [15]. The respective I3/I1 values have been plotted as a function of polymer concentration in Fig. 5b and the CMC of polymer a-CF is determined to be 20 mg/L. This is a reasonably low concentration, suggesting that micelles fabricated out of polymer aCF can remain stable in solution, even after extreme dilution. This is indeed the case that will be experienced upon intravenous injection into the much larger volume of blood for systemic circulation. Further, it has to be reminded that this study was done in DI water. In an

k, l

7.6

3.4. Micellar size and morphology In order to achieve longevity during systemic circulation, the micelles must be small enough to evade detection and destruction by the reticulo-endothelial system. Table 2 summarizes the average particle size and polydispersity of blank and paclitaxel-loaded polymer a-CF micelles done in three different pH. All blank micelles are safely below 180 nm, with an acceptably good polydispersity index between 0.28–0.36. The slight increase in size of the micelles from about 160 nm at the lower pH of 5 and 6, to about 176 nm at pH 7.4, however, can be attributed to the deprotonation (pKa 6.8 [7]) of the –COOH groups of UA

m, n

7.4

7.2

7.0

6.8

m, n

k, l

8

ionic solution, such as our blood, the polymer is expected to exhibit an even lower CMC value due to the ‘‘salting out’’ effect [16].

7

6

5

4

3

2

1

ppm Fig. 4. 1H NMR spectrum of polymer a-CF in D2O. Sharp superimposed peak at d 2.6 ppm is most likely due to DMSO (C2H5SO), a solvent used extensively during synthesis. Inset is a blown up of the main spectrum from d 6.60 to 7.60 to show folate peaks.

I1 = 373 3

I3 = 393 Increasing polymer

2.2

concentration (g/L)

8.00 x 10-2 4.00 x 10-3 6.00 x 10-4 7.50 x 10-5 1.00 x 10-5

2

b

1

CMC ~ 20 mg/L

2.0 1.8 I3 / I1

Arbitrary Intensity (x100000)

a

1.6 1.4 1.2 1.0

0 360

380 400 420 Wavelength (nm)

440

0.8 -2.0

-1.0

0.0 1.0 lg (mg/L)

2.0

3.0

Fig. 5. (a) Emission spectrum recorded for pyrene equilibrated with polymer a-CF in DI water at various polymer concentrations. Pyrene concentration: 6.16  107 M. (b) I3/I1 plotted as a function of polymer concentration. CMC: 20 mg/L.

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Table 2 Average size and polydispersity index (PI) of blank and paclitaxel-loaded polymer a-CF micelles Micelle size

pH 5.0

pH 6.0

pH 7.4

In DI, drug loaded

Polymer a-CF

15972 nm (0.32)

15972 nm (0.32)

17673 nm (0.36)

22272 nm (0.29)

Fig. 6. A SEM micrograph of paclitaxel-loaded micelles.

to become –COO at a higher pH. The electrostatic charge repulsion experienced between chains then diametrically expands the spherical micelles and result in a larger size. The hydrodynamic particle size of the drug-loaded micelles (220 nm, Table 2) is understandably larger than the blank micelles, probably due to the incorporation of large and bulky drug molecules (Mw of paclitaxel: 853.9 g/mol) within the core. The particle size and polydispersity observed in the SEM image (Fig. 6) of this same batch of drug-loaded micelles also agrees reasonably well with the values obtained from light-scattering measurements. 3.5. pH-induced thermo-sensitivity PNIPAAm exhibits a LCST of around 32 1C and phase separates out of water from its soluble state (or vice versa) in a predictable fashion at that temperature [17]. This unique temperature sensitivity of PNIPAAm is widely studied and has been suggested to hinge on a critical hydrophilic/phobic balance between the chemical groups on the polymer. DMAAm, with its shorter hydrocarbon chain is more hydrophilic than NIPAAm and has been added to increase the LCST of the overall copolymer. From Table 3, it can be clearly observed that micelles fabricated out of the bare backbone polymer indeed show a

trend of increasing LCST with increasing DMAAm content. Further, this trend is consistent at all three pH values tested. UA, with its long hydrocarbon chain is much more hydrophobic than either NIPAAm or DMAAm. It has thus been added to confer an amphiphilic nature to the polymer, and to impart pH sensitivity to the overall polymer by virtue of its ionizable –COOH group. From Table 4, it is clear that this pH sensitivity has been achieved by polymer a-CF such that it has a LCST of 37.8 1C (normal body temperature: 37 1C) at the normal physiological pH of 7.4 and a depressed LCST of 32.8 and 34.8 1C at the more acidic pH of 5.0 and 6.0, respectively. This suggests that micelles made out of polymer a-CF can safely encapsulate its drug content and remain well soluble during systemic circulation. On the other hand, the core-shell structure will be deformed and drug is preferentially released at acidic tumor sites or in the endosomes/ lysosomes where pH is typically lower (extracellular pH of tumor cells: pH 7.0 or lower, endosomes: pH 5.0–6.5 and lysosomes: pH 4.0–5.5). As listed in Table 4, it can also be seen that the polymer experiences a LCST drop upon cholesterol grafting due to the addition of a much more hydrophobic group to the chain. Folate, on the other hand, is relatively more hydrophilic than cholesterol. However, its hydrophilicity still pales in comparison to the remaining sections of the chain. Its addition thus has little effect on the overall LCST. This then accounts for the similarities in LCST observed for polymers a-C and a-CF. To study for the effects of ionic strength, buffers at a higher strength of 0.167 M were prepared. As observed in Table 4, an increase in ionic strength depresses the LCST observed, as similarly reported in the literature [18]. This can be explained as due to the extra ionic competition for bonding water with the hydrophilic groups on the polymer, resulting in a reduced extent of H-bonding with water molecules. The polymer chains, being less immobilized by water molecules, therefore, precipitate at a lower temperature. 3.6. Drug loading level The drug loading level of polymer a-CF micelles fabricated under the conditions described earlier was determined to be about 2.6% in weight. The large size of the paclitaxel molecule could be one reason for the modest loading level observed. Results also indicate that increasing the initial amount of paclitaxel during the fabrication process does not improve the final loading level. In this

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Table 3 Effects of pH and polymer composition on LCST (1C) Polymer sample

pH 5.0

pH 6.0

pH 7.4

Polymer a Polymer b Polymer c

33.3 33.7 36.1

35.3 35.8 37.3

38.8 39.5 40.1

Table 4 LCST values (1C) of polymer a, a-C and a-CF as a function of pH and effect of ionic strength on LCST of polymer a-CF Polymer (ionic strength of buffer)

pH 5.0

pH 6.0

pH 7.4

a (0.154 M) a-C (0.154 M) a-CF (0.154 M) a-CF (0.167 M)

33.3 32.7 32.8 32.3

35.7 35.0 34.8 34.3

38.8 37.6 37.8 37.2

3.7. In vitro paclitaxel release The in vitro release profiles of paclitaxel-loaded polymer a-CF micelles have been obtained at 37 1C for pH 7.4 (to simulate the extracellular pH of normal healthy tissues) and pH 5.0 (modelled after the acidic endosomal or lysosomal environment of cells) (Fig. 7). As can be seen, drug release is pH-sensitive. For example, the release of paclitaxel at pH 7.4 is slow and sustained, with only 8% of the total drug content being released within the first 5 h. This suggests that the amount of unintended drug lost from the micelles during systemic circulation is relatively suppressed. Drug release at pH 5.0 is, however, much faster with close to 21% of its total drug content being

70 Cumulative drug release (%)

case, doubling the initial amount of drug used from 1 to 2 mg resulted in a loading level of 2.5 wt%. This may mean that doing so, instead of improving the final drug loading, may cause drug precipitation during fabrication and also a greater diffusional loss to the external sink during dialysis. On top of this, other parameters—for example, the dialysis temperature and duration, pore size of membrane, polymer to drug ratio and organic solvent chosen—can potentially influence the final drug loading level. This, therefore, points to a need to further optimize the conditions used during drug loading in order to increase drug content. Nonetheless, even at this modest loading level, the solubility of paclitaxel has already been successfully increased to many times its intrinsic water solubility limit. For instance, 2.1 mg of the freeze-dried drug-loaded micelles was easily dissolved in 2 mL of DI water in this study (higher concentrations might be possible). At a loading level of 2.6 wt%, the effective concentration of paclitaxel would be 27.3 mg/L, which is already 91 times higher than its intrinsic water solubility of 0.3 mg/L. This observation further justifies the actual existence of the coreshell architecture, without which, such a high concentration of paclitaxel in water would have been unachievable.

60 50 40 30 20 10 0 0

12

24

36

48

60

72

Time (hours) Fig. 7. In vitro release profile of polymer a-CF loaded with paclitaxel at pH 7.4 (m, normal physiological pH) and 5.0 (’, acidic endosomal and lysosomal pH) at 37.070.1 1C.

released within the first 4 h and nearly 44% by the first 24 h. At pH 5.0, the LCST of polymer a-CF micelles is below 37 1C (32.8 1C), causing it to precipitate out of the solution. With the deformation of the core-shell structure, an accelerated release rate is observed. Polymer precipitation is visually confirmed by the observation of white precipitates in the dialysis bag within the first 5 h. On the other hand, the LCST of the micelles at pH 7.4 is above 37 1C (37.8 1C). As such, the polymer remains well soluble within the solution and the drug is safely encapsulated. Drug leached from the micelles is then attributed to an inevitable out-diffusion process through the core-shell structure. Therefore, a generally smooth and constant release profile is exhibited by the micelles at pH 7.4. 3.8. In vitro cytotoxicity assay against KB cells The potency of paclitaxel derives from its ability to catalyze the polymerization of tubulin dimmers into

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stabilized microtubules and hence, inhibit cell mitosis in their late G2 or M phases [1]. To achieve therapeutic effects, the drug must first be successfully internalized via endocytosis, before escaping into the cytoplasm. Clearly, a drug or its carrier delivered to the surface exterior of the cell is not guaranteed of reaching its intended site of action as there are several biological barriers (e.g., the cell and endosomal membranes) which first have to be overcome. The relative cytotoxicity of drug-loaded polymer a-C, polymer a-CF and the pure drug is shown in Fig. 8. Increasing drug concentration reduces cell viability. The IC50 value of pure paclitaxel is about 0.12 mg/L, which is clearly a low concentration and serves to demonstrate the potency of the drug. Paclitaxel-loaded polymer a-C

100 Pure Paclitaxel (IC50 = 0.120 mg/L) Polymer a-C (IC50 = 0.115 mg/L)

Cell viability (%)

80

Polymer a-CF (IC50 = 0.045 mg/L)

60

40

20 0

0.1 0.2 0.3 Paclitaxel concentration (mg/L)

0.4

0.5

Fig. 8. Viability of KB cells after exposure to pure paclitaxel, paclitaxelloaded polymer a-C and polymer a-CF micelles at various drug concentrations at 37 1C for 48 h.

a

micelles induce similar cytotoxicity when compared to the pure paclitaxel, demonstrating that the triggering mechanism for the release of drug from the endosomes/lysosomes into the cytosols is feasible and according to design. In sharp contrast, paclitaxel-loaded polymer a-CF micelles are much more toxic against KB cells, compared to either pure paclitaxel or paclitaxel-loaded polymer a-C micelles. This is reflected by the corresponding IC50 value, which is about two times lower. This suggests that the cellular uptake efficiency of these micelles is greatly enhanced, most probably due to a folate receptor-assisted endocytosis mechanism. Similarly, it is believed that the internalized micelles then release their drug content from the endosomal/lysosomal environment into the cytosol. In order to further evaluate the role of folate in the cellular uptake of paclitaxel-loaded polymer a-CF micelles, the cells are exposed to a constant drug concentration of 0.15 mg/L while being maintained in growth media supplemented with increasing concentrations of free folate. It is found that cell viability increases with increasing folate concentration (Fig. 9). For instance, the cell viability is 34% in the absence of folate but increased to 53% in the presence of 500 mg/L free folate. These results suggest that free folate molecules hamper the cellular uptake of the micelles by competitive binding to the folate receptors on the cell surfaces. Interestingly, it has been suggested that eating a folate-restricted diet may improve the in vivo therapeutic efficiencies of such folate-conjugated carriers [19]. Fig. 9b proves that no significant cytotoxicty effects are associated with the blank polymer up to a concentration of 20 mg/L. At a loading level of 2.6 wt%, an effective drug concentration of 0.5 mg/L corresponds to a polymer concentration of 20 mg/L. Therefore, it confirms the proposal that the micelles act merely as vehicles for the drug. Any cytotoxicity observed is thus mainly attributed to the effects of the released drug alone.

b

60

1739

120

50

Cell viability (%)

Cell viability (%)

100

40

80 60 40 20

30

0

100 200 300 400 Folate concentration (mg/L)

500

0

0 4 8 12 16 20 Polymer concentration (mg/L)

Fig. 9. (a) Effect of free folic acid on viability of KB cells incubated with paclitaxel-loaded folate-conjugated polymer a-CF micelles at paclitaxel concentration of 0.15 mg/L. (b) Viability of KB cells when exposed to varying concentration of blank polymer a-CF micelles.

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4. Conclusion Folate-conjugated P(NIPAAm-co-DMAAm-co-UA)-gcholesterol has been successfully synthesized and utilized to encapsulate a highly hydrophobic drug, paclitaxel. The micelles expose a shell decorated with folate that can specifically recognize cancer cells, which overexpress their surface folate receptors. The water-solubility of paclitaxel increased significantly. The particle size and LCST of the drug-loaded micelles were responsive to the external pH values. Therefore, paclitaxel release was also pH-sensitive, being more rapid in an acidic environment. This suggests that drug release can be triggered within the acidic intracellular environments. Paclitaxel-loaded polymer aCF micelles are the most effective in killing KB cells due to an enhanced folate receptor-mediated endocytosis process. No significant cytotoxicity, however, is observed with the blank polymeric carriers themselves. This polymeric vehicle is, therefore, anticipated to be a promising paclitaxel carrier for its targeted and intracellular delivery. Acknowledgments This work was funded by Institute of Bioengineering and Nanotechnology, and Singapore Imaging Consortium (Grant No.: 005/2005), Agency for Science, Technology and Research, Singapore. References [1] Panchagnula R. Pharmaceutical aspects of Paclitaxel. Int J Pharm 1998;172:1–15. [2] Muller RH, Jacobs C, Kayser O. Nanosuspensions as particulate drug formulations in therapy rationale for development and what we can expect for the future. Adv Drug Deliv Rev 2001;47:3–19. [3] Kim SC, Yoon HJ, Lee JW, Yu J, Park ES, Chi SC. Investigation of the release behavior of DEHP from infusion sets by paclitaxel-loaded polymeric micelles. Int J Pharm 2005;293:303–10. [4] Wang J, Mongayt D, Torchilin VP. Polymeric micelles for delivery of poorly soluble drugs: preparation and anticancer activity in vitro of paclitaxel incorporated into mixed micelles based on poly(ethylene glycol)-lipid conjugate and positively charged lipids. J Drug Target 2005;13:73–80.

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