Minimalism in fabrication of self-organized nanogels holding both anti-cancer drug and targeting moiety

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Colloids and Surfaces B: Biointerfaces 63 (2008) 55–63

Minimalism in fabrication of self-organized nanogels holding both anti-cancer drug and targeting moiety Sungwon Kim a,1 , Kyong Mi Park b,1 , Jin Young Ko a , Ick Chan Kwon a , Hyeon Geun Cho c , Dongmin Kang d , In Tag Yu e , Kwangmeyung Kim a,∗ , Kun Na b,∗∗ a

Biomedical Research Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791, Republic of Korea b Department of Biotechnology, The Catholic University of Korea, 43-1 Yokgok-dong, Wonmi-gu, Bucheon 420-743, Republic of Korea c Department of Internal Medicine, Kwandong University, 697-24 Hwajeong-dong, Deogyang-gu, Gyeonggi-do 412-270, Republic of Korea d Division of Life and Pharmaceutical Sciences, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-gu, Seoul 120-750, Republic of Korea e Korea Basic Science Institute, Chuncheon Center, 192-1 Hyoja2-dong, Chuncheon, Kangwon-do 200-701, Republic of Korea Received 1 October 2007; accepted 11 November 2007 Available online 22 November 2007

Abstract Recent researches to develop nano-carrier systems in anti-cancer drug delivery have focused on more complicated design to improve therapeutic efficacy and to reduce side effects. Although such efforts have great impact to biomedical science and engineering, the complexity has been a huddle because of clinical and economic problems. In order to overcome the problems, a simplest strategy to fabricate nano-carriers to deliver doxorubicin (DOX) was proposed in the present study. Two significant subjects (i) formation of nanoparticles loading and releasing DOX and (ii) binding specificity of them to cells, were examined. Folic acid (FA) was directly coupled with pullulan (Pul) backbone by ester linkage (FA/Pul conjugate) and the degree of substitution (DS) was varied, which were confirmed by 1 H NMR and UV spectrophotometry. Light scattering results revealed that the nanogels possessed two major size distributions around 70 and 270 nm in an aqueous solution. Their critical aggregation concentrations (CACs) were less than 10 ␮g/mL, which are lower than general critical micelle concentrations (CMCs) of low-molecular-weight surfactants. Transmission electron microscopy (TEM) images showed well-dispersed nanogel morphology in a dried state. Depending on the DS, the nanogels showed different DOX-loading and releasing profiles. The DOX release rate from FA8/Pul (with the highest DS) for 24 h was slower than that from FA4/or FA6/Pul, indicating that the FA worked as a hydrophobic moiety for drug holding. Cellular uptake of the nanogels (KB cells) was also monitored by confocal microscopy. All nanogels were internalized regardless of the DS of FA. Based on the results, the objectives of this study, to suggest a new method overcoming the complications in the drug carrier design, were successfully verified. © 2007 Elsevier B.V. All rights reserved. Keywords: Folate; Pullulan; Self-organized; Nanogels; Doxorubicin; Cancer

1. Introduction Self-organized nanogels of hydrophobically modified polysaccharides have been intensively investigated in the biomedical and pharmaceutical fields due to their potential based on biocompatibility and abundance [1–4]. It is well known that nanogels consist of polycore and hydrophilic shell to make zero Gibb’s energy and are able to enclose a water-insoluble drug in

∗

Corresponding author. Tel.: +82 2 958 5912; fax: +82 2 958 5909. Corresponding author. Tel.: +82 2 2164 4832; fax: +82 2 2164 4865. E-mail addresses: [email protected] (K. Kim), [email protected] (K. Na). 1 These authors equally contributed to this work. ∗∗

0927-7765/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2007.11.009

polycore and to protect interactions with surrounding biological materials. Thus, nanogels have high stability in aqueous solution [5]. In addition, drugs loaded in the particle’s hydrophobic core usually enhance the stability of self-organized nanogels, in view of ‘like dissolves like’ principle [6]. Drug-loaded nano-carriers can be used for cancer therapy without any targeting moieties. In this case, the therapeutic effect can be achieved by a specific pathophysiology of cancer, named as enhanced permeability and retention (EPR) effect [4,6]. Since the malignant neoplasms grow faster than the normal tissues, they necessarily require much of nutrients and oxygen supply, which results in formation of new blood vessels around the tumor tissues. Due to the fast growth, the angiogenic vessels have leaky pores that are large enough for nano-carriers

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to escape out of vessel. Thereafter, nano-materials as well as macromolecules can be accumulated into the cancer tissue. On the other hand, introduction of targeting moieties to nanocarriers can endows more specificity to the target tissues. To achieve active targeting like this, famous compounds including folate [7], transferrin [8], or monoclonal antibodies [9] have been conjugated to the amphiphilic polymers. Although targeting moieties to tumor cells increase therapeutic efficacy due to the specificity, amphiphilic polymers must be further modified to introduce such molecules via more chemical reaction steps. In addition, it is considerable that there are no reports about if the conjugation of targeting compounds has an influence on the physicochemical property of polymers or nano-carriers, even though most of such molecules possess high hydrophobicity or large molecular weight. Therefore, in this study, we propose a simplest method to fabricate self-organized nanogels consisting of folic acid (FA) and pullulan. Pullulan, a homopolysaccharide, has been an attractive material in the field of pharmaceutics because of its low immuogenecity and biodegradability [10,11]. Moreover, pullulan is likely to be dissolved in an organic solvent, dimethyl sulfoxide (DMSO), as we reported previously [12]. Since anhydrous condition is critical to achieve a high yield in a coupling reaction, such solubility is good for further chemical modification of the pullulan. We hypothesized that, since FA possesses very low solubility in aqueous solution at neutral pH (1.6 ␮g/mL at 25 ◦ C), it was expected that conjugation of FA to the pullulan using the simple one-step chemistry possibly fabricated a new kind of nanoparticle. Two major subjects focused in the present research are (i) whether nano-scale self-organized nanogels could be formed from such a simple method, and (ii) whether the nanogels still seized specificity to bind to target cells. In addition, the feasibility of the nanogels loading anticancer reagent, doxorubicin, to treat cancer was also examined by drug release and toxicity tests. 2. Materials and methods 2.1. Materials Folic acid, doxorubicin (DOX), fluorescein isothiocyanate (FITC), 37% formaldehyde solution, 1,3-dicyclohexyl carbodiimide (DCC), 4-dimethylaminopyridine (DMAP), triethylamine (TEA), dimethyl sulfoxide, N,N-dimethyl acetamide (DMAc), phosphorous pentoxide (P2 O5 ), 4-(2-hydroxyethyl)1-piperazine ethansulfonic acid (HEPES), sodium bicarbonate, glutaraldehyde, dibutyltin dilaurate, RPMI1640 medium and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Sigma–Aldrich Corp. (St. Louis, MO). Fetal bovine serum (FBS), antibiotics (penicillin/streptomycin), and Dulbecco’s phosphate buffer saline (DPBS) were obtained from GibcoBRL (Invitrogen Corp., Carlsbad, CA). Since the FA contained 8.1% (w/w) moisture, it was purified as described next: After dissolved in 200 mL DMSO (1%, w/v) by continuous stirring at 40 ◦ C, the FA was precipitated against 800-mL deionized water. The solution was further acidified down to pH 4.0 by adding 2N HCl solution, which led to

a yellow slurry. Solid component was obtained by filtration and dissolved in 2N NaOH solution. After repeatedly washing with distilled water and removing insoluble particles, the solution was acidified again (pH ≈4.5). The precipitant was collected by filtration and, with P2 O5 , dried under vacuum at 40 ◦ C for 2 days (Yield; 88%). Pullulan (Pul) was obtained from Hayashibara (MW 200,000 Da, Okayama, Japan), which was purified by the following method. At first, the pullulan was dissolved in 300 mL double distilled water (2%, w/v), and the solution was slowly dropped into 700 mL ethyl alcohol. White precipitant was collected and dried under vacuum condition for a day. The dried pullulan was dissolved in 300-mL deionized water again, and successively filtrated through filter membranes (Millipore Corp., Billerica, MA) with 5, 0.8, and 0.2 ␮m pore sizes. Final product was acquired by lyphilization of the filtrate and drying under vacuum with P2 O5 at RT (Yield; 96%). 2.2. Conjugation of folate to pullulan Three FA/Pul conjugates with different degree of substitutions (DSs) were synthesized as illustrated in Fig. 1. Purified Pul (1 g, 6.173 mmol of repeating glucose units) was dissolved in 30 mL anhydrous DMSO by stirring at 60 ◦ C for 20 min followed by adding determined amount of FA and DMAP (247, 370, and 494 ␮mol for 4, 6, and 8% DS, respectively, and according to the initial feed ratio of FA, each FA/Pul was named as FA4/Pul, FA6/Pul, and FA8/Pul.). Overnight stirring at room temperature was required to clearly dissolve the FA. After adding slightly excess amount of DCC into each flask, reaction was carried for 2 days at 25 ◦ C. After filtration with 0.45 ␮m filter membrane, the solutions were directly put into dialysis membrane (MWCO 12,000–14,000 Da, Spectra/Por RC dialysis membrane, Spectrum Laboratories, Inc., Rancho Dominguez, CA) and dialyzed against excess deionized water for 1 day at room temperature and for another 4 days at 4 ◦ C. Final products of FA/Pul conjugates were obtained by freeze- and vacuum-drying. All procedures were performed under light-protected condition and each FA/Pul was stored at −20 ◦ C for further experiments (Yields; 89–93%). Chemical conjugation was confirmed by 1 H NMR spectroscopy and the degree of substitution was determined by UV–vis spectrophotometry (Model Lamda 18, PerkinElmer Inc., Wellesley, MA). After dissolving FA/Puls and FA in DMSO at various concentrations, absorbance spectra ranged from 190 to 450 nm were scanned, which showed three λmax s at 320, 360, and 380 nm. Among those peaks, absorption values of the FA solution with different concentrations at 320 nm gave a good plot of the linear regression. Using the standard curve, the extinction coefficient (ε) could be calculated, that was similar to a value addressed in another report. Fluorophore labeling of FA/Puls was accomplished by mixing 100 mg FA/Pul with 0.5 mg FITC in 10 mL anhydrous DMSO containing anhydrous 1 ␮L dibutyltin dilaurate. After reaction for 18 h at RT, the solution was dialyzed (MWCO 12,000–14,000 Da, Spectra/Por) against excess deionized water for 5 days at 4 ◦ C, followed by lyophilization. Since the quantity of FITC was too small to modify physicochemical property of

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Fig. 1. Synthetic scheme of folate-pullulan conjugates (FA/Puls).

FA/Pul, each FITC-labeled FA/Pul was used for other experiments without further characterization. 2.3. Preparation and characterization of folate/pullulan nanoparticles To fabricate self-organized nanogels, each FA/Pul (100 mg) was dissolved in 10 mL DMSO and dialyzed (MWCO 3500 Da, Spectra/Por membrane RC) against excess deionized water at dark cold room. In 3 days, dialysates were collected and the volume of them was adjusted to 100 mL (final concentration 1 mg/mL). After filtration of each sample with 0.8 ␮m syringe filter, particle size and its distribution were measured by dynamic light scattering (argon ion laser system, Laxel Laser model 95, Brookhaven Instrument Corp., Holtsville, NY). Transmission electron microscopy (TEM, Philips CM 30, Philips Electron Optics, Eindhoven, Netherlands) was also conducted at the accelerating voltage 200 keV in order to observe the gels in a dried state. 2.4. Measurement of critical aggregation concentration (CAC) The CACs of the conjugates was calculated by using fluorescence spectroscopy (pyrene). Pyrene stock solution (6.0 × 10−2 M) was prepared in acetone and stored at 5 ◦ C until used. For the measurement of steady-state fluorescence spectra, the pyrene solution in acetone was added to distilled water to give a pyrene concentration of 12.0 × 10−7 M. The solution was then distilled under vacuum at 60 ◦ C for 1 h to remove acetone from the solution. The acetone-free pyrene solution was mixed together with solutions of self-organized nanogels of which the concentration ranged from 1 × 10−5 to 1.0 mg/mL.

The final concentration of pyrene in each sample solution was 6.0 × 10−7 M, which is nearly equal to its solubility in water at 25 ◦ C [2]. 2.5. Anti-cancer drug loading and release DOX (doxorubicin, 10 mg) was dissolved in anhydrous 10 mL of DMSO containing 6.2 ␮L TEA. After adding each FA/Pul conjugate (50 mg) into the solution, the mixture was stirred overnight at dark cold room. To produce DOX-loaded FA/Pul nanogels, the solution was transferred into wet dialysis membrane (Spectra/Por RC dialysis membrane, MWCO 1000 Da) and dialyzed against excess deionized water at RT. Water was refreshed every 8 h and, in 24 h, the dialysate was filtrated (0.45 membrane filter, Millipore) to remove aggregated precipitants. Finally, the filtrate was lyophilized for 3 days and then, the products were kept at −20 ◦ C till further experiments. To determine the drug-loading content, the dried samples were suspended in DMAc and vigorously stirred for 2 h. After brief sonication (3 min), the solution was centrifuged at 20,000 × g for 30 min. UV absorbance of the supernatant containing DOX extracted from nanogels was measured at 490 nm. With using a linear standard curve out of free DOX in DMAC, the loading efficiency could be calculated, which were 12 ± 5.4, 16 ± 6.8, and 20 ± 4.9% (w/w) for FA4/, FA6/, and FA8/Pul, respectively. In vitro drug release test was conducted by using dialysis tube. After entrapping 2 mL DOX-loaded FA/Pul nanogels (0.1 mg/mL DOX content), dialysis tubing was placed in a vial containing 10 mL sterile PBS (pH 7.4) and mildly stirred (100 rpm) at 37 ◦ C. At a certain time, whole medium was taken and kept at −4 ◦ C, and fresh PBS was added without

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disrupting the sink condition. Every sample medium containing released DOX was analyzed by UV spectrophotometry as described above, which resulted in a release plot as a function of time.

samples were snapped with an exposure time 200 ms. Obtained images did not undergo further adjustment process except resizing and making montages by means of NIH Image J, version 1.33u.

2.6. Cell culture

2.9. Competition between FA/Pul and free FA

KB cell was obtained from Korea Cell Line Bank (KCLB No. 10017 and 10061, respectively). KB (passage 9) was cultured, respectively, in RPMI 1640 with supplement of 10% heatinactivated FBS, 1% penicillin/streptomycin, 5 mM sodium bicarbonate, and 20 mM HEPES under humidified air containing 5% CO2 at 37 ◦ C. Before experiments, cell was sub-cultured in 100 mm culture dish and then, at 70–80% confluence state, placed into other culture plates (1:4 ratio).

Specific interaction of FA/Pul with cells expressing FA receptors (FARs) was conducted by a competition study. After adding 1 mg/mL FA8/Pul into 6-well culture plate containing KB cell in 1 mL KRH (w/0.1% BSA, pH 7.4), FA solution in PBS with different concentrations (0.05, 0.1, and 0.5 mg/mL) was treated simultaneously. In 2 h, cells were rinsed twice with PBS and applied by 1 mL fixing solution. Fluorescence microscopy was conducted as described above.

2.7. Toxicity test

2.10. Statistics

To examine the anti-cancer effect of DOX loaded in nanogels, MTT assay was carried out. KB cell was seeded in 24-well culture plate (105 /well) and cultured in 1 mL culture media for a day. After each well was rinsed twice by 1 mL pre-warmed media without FBS, 1 mL medium containing 10% FBS was added and then, free DOX or DOX-loaded FA/Pul nanogels (50 ␮L PBS, 5 ␮g DOX equivalent) were treated. PBS and FA/Pul nanogels without loading DOX were used for controls. In 0.5, 1, 2, and 3 days, media was removed and cells were washed with warm FBS-free medium, followed by addition of 500 ␮L MTT solution (0.1% in KRH). After 12 h incubation, cells were rinsed by PBS and formazan crystal inside cells was dissolved by 150 ␮L DMSO. By measuring the optical density at 590 nm (OD590 ), viability was acquired.

Every experiment was carried out at least in triplication and acquired data were expressed as mean ± standard deviation (S.D.). In UV absorption tests, a simple model of linear regression (y = ax + b) was used, and the a value when the b value was close enough to zero was selected as an extinction coefficient. Graphs from release test were analyzed by an exponential rise model equation, y = y0 + a(1 − e−bx ). Viability from the MTT assay was analyzed by Student’s unpaired t-test and statistical significance of each value in comparison with the control level at a given time was marked as both asterisk (* P < 0.01) and cross (+ P < 0.05). 3. Results 3.1. Conjugation of folate to pullulan

2.8. Cell binding and uptake of nanogels Cellular uptake of either free or DOX-loaded nanogels was investigated using fluorescence microscope (Karl Zeiss, Oberkochen, Germany). At first, KB cell (105 /well) was seeded into 6-well cell culture plate and incubated in 2 mL FBSand antibiotics-supplemented media for a day. The cell was rinsed twice with warm PBS and embedded in 1.8 mL Kreb’s Ringer HEPES (KRH) buffer (4.74 mM KCl, 1.19 mM KH2 PO4 , 1.19 mM MgCl2 , 119 mM NaCl, 2.54 mM CaCl2 , 25 mM NaHCO3 , 10 mM HEPES, 1 g/L d-glucose, and 0.1% BSA, pH 7.4). To examine whether the DS of FA had influence on binding property of the nanogels onto cells, the same amount of FITC-labeled FA4/, FA6/, and FA8/Pul was treated to each well. In this experiment, the amount of FITC-labeled FA/Pul was 1 mg/mL regardless of the DOX-loading efficiency. Before adding nanogels, each well was washed twice with warm PBS. After 1-h incubation at 37 ◦ C, the cell was washed again with PBS and, at RT for 5 min, fixed by 1 mL of 2% glutaraldehyde and 2% formaldehyde in PBS (fixing solution). Every sample was stored at 4 ◦ C till performing fluorescence microscopy. To observe fluorescence images, more PBS (2 mL) was added into each well. Every sample was triplicated and five different images per well were acquired. Using water-emerging lens (×400),

Chemical conjugation was accomplished by a conventional carbodiimide reaction. It is well known that carbonyl compounds preferably bind to hydroxyl groups at C(6) of a polysaccharide. The pullulan is representative polysaccharide that is readily dissolved in organic solvent. Such solubility makes it easy to be chemically modified. 1 H NMR spectra showed that the FA was successfully conjugated to pullulan. 1 H NMR (300 MHz, DMSO-d6 ) presented δ 2.29 (t, 2H, COCH2 ), 4.27 (q, 2H, CH2 CH), 6.9 (t, 1H, CHCO), 7.64 (s, 2H, COCH = CH), 6.64 (s, 2H, COCH = CH), 8.10 (s, 2H, CH2 C) for the folic acid and broad multiple peaks in the range of 3–6 ppm for the pullulan. In the UV experiment, the molar extinction coefficient (ε) of FA was 2266 M−1 cm−1 calculated from a standard curve, y = 4.3485x − 0.0501 (R2 = 0.9978). Calculated degree of substitution for each FA/Pul (the number of FAs per hundred glucose units in pullulan) was 3.29, 5.61, and 8.14. 3.2. Preparation of nanogels Fig. 2 displays the particle size and its distribution of each FA/Pul nanogel in an aqueous environment (Fig. 2a FA4/, Fig. 2b FA6/, and Fig. 2c FA8/Pul). Two major peaks and

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Fig. 2. Size distribution of FA/Pul nanogels determined by dynamic light scattering. Two major groups in size distribution were detected in the range of 60–80 and 230–300 nm. Minor peaks around 1000 nm completely disappeared as the DS of FA increased. At the same time, % intensity of small particles around 70 nm also became lowered.

one minor peak were detected in each FA/Pul sample. Dominant particle size was about 230–300 nm (mean values of 261, 276, and 272 nm for FA4/, FA6/, and FA8/Pul, respectively), and particles with another major size were distributed in a range of 60–80 nm (71, 75, and 74 nm in the same order). Size and its distribution of particles in those ranges were not changed regardless of the DS of FA. On the other hand, particles appearing around 1000 nm became disappeared as the DS increased (6, 2, and 0% intensity in Fig. 2a–c, respectively). To define the threshold polymer concentration necessary for the self-assembly of nanogels through intra- or intermolecular associations, their critical aggregation concentration (CACs) were determined by monitoring the change in the intensity ratio (I338 :I335 ) of the pyrene. The calculated CACs were 9.98, 7.4, and 2.6 ␮g/mL for FA4/, FA6/, and FA8/Pul, respectively. The nanogels from FA/Pul conjugates were also highly stable in a dried state, which was confirmed by TEM images (Fig. 3a FA4/, Fig. 3b FA6/, and Fig. 3c FA8/Pul). However, the image of FA4/Pul shows many small aggregates, which possibly resulted in further aggregation of each nanoparticle in aqueous medium as shown in Fig. 2. Population of such small particulates became decreased as the DS of FA or the hydrophobicity of polymer more increased. Overall size of nanoparticle in the TEM snaps was less than 50 nm because water from samples was eliminated. On combining the light scattering results, shrinking

Fig. 4. Doxorubicin (DOX) release from FA/Pul nanogels (circle 䊉, FA8/Pul, triangle , FA6/Pul, and square , FA4/Pul). Higher content of FA released DOX more slowly. Data are expressed as mean ± S.D. (n = 3).

their size reflects that the particles possess huge hydrodynamic volume. 3.3. Anti-tumor drug release In vitro release profile of DOX from FA/Pul nanogels was examined and the result was displayed in Fig. 4. Regression

Fig. 3. TEM images of FA/Puls in a dried state ((a) FA4/, (b) FA6/, and (c) FA8/Pul). Many small aggregates (<20 nm) were detected in FA4/Pul, but became integrated in bigger particles (c.a. 50 nm) as the DS of FA increased. Scale bar below each image represents 500 nm (a and b) or 100 nm (c).

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Fig. 5. Cytotoxicity test in terms of the KB cell viability determined by MTT assay. All results from samples treated by 5 ␮g/mL DOX were normalized by the optical density at 590 nm of control (PBS) and expressed as mean ± S.D. of % viability (n = 3).

Fig. 6. Fluorescence microscopic images to examine the effect of DS of FA on cellular binding and uptake of DOX-loaded FA/Pul nanogels. FITC channel for polymer (green) and RITC channel for DOX (red) are simultaneously presented (FA4/, FA6/, and FA8/Pul from the top). No significant difference is detected depending on DS of FA.

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equation for each group was y = 3.133 + 92.9794(1 − e−0.0875x ), y = 0.8497 + 93.7875(1 − e−0.0584x ), and y = 2.4669 + 93.2957 (1 − e−0.0367x ) for FA4/, FA6/, and FA8/Pol, respectively. Each equation showed good correlation factor in that R2 values were 0.9987, 0.9985, and 0.9987. Half-maximal release time (t1/2 ) that freed 50% of loaded DOX from nanogels depended on the DS of FA, which was 8.0, 12. 7, and 19.4 h for FA4/, FA6/, and FA8/Pul, respectively. As FA substitution increased, t1/2 was prolonged. In other words, release rate of DOX from the nanogels was more accelerated as the DS decreased. The FA4/Pul fabricated about 0.13 mg DOX (63% of total 0.2 mg), while FA8/Pul did only 37%, within just 12 h. In 3 days, total amount of released DOX reached up to 96, 93, and 89% for each nanoparticle in that order. Saturation point for each nanoparticle was around 95–96%, which meant that almost all drugs were liberated from each nanoparticle within 3–4 days.

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3.4. Cytotoxicity test Co-incubation of cells with DOX-loaded nanogels presented therapeutic effect of the drug carriers. Fig. 5 displays viability of KB cells in comparison with control (PBS). Viability of KB cells was significantly decreased (P < 0.01 from Student’s t-test for all) by challenging free DOX. Nanogels without loading DOX did not gain any statistical significance, which meant that, regardless of the incubation time, there was no cytotoxicity induced by nanogels. On the other hand, results from FA/Pul nanogels containing DOX present that the cell viability was significantly declined at least after day 1. 3.5. Specific interaction of FA/Pul nanogels with cells Fig. 6 shows that the effect of DS on cellular uptake of nanoparticle is negligible, since no difference in fluorescence

Fig. 7. Competition between free FA and DOX-loaded FA8/Pul nanogels. FITC channel for polymer (green) and RITC channel for DOX (red) are simultaneously presented (0.5, 0.1, and 0.05 mg/mL FA from the top). It is obvious that, as the concentration of free FA increases, binding and uptake of nanogel decreased more.

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intensity between each group is detected. In 1 h after challenging DOX-loaded nanogels, most of nanogels were detected inside cytosol of KB cells. FITC channel displays the FITC-labeled FA/Pul and RITC (rhodamine B isothiocyanate) channel shows the DOX fluorescence. To confirm whether the difference was due to the folate receptors, competition study was carried out and expressed in Fig. 7. It was revealed that free folic acid effectively competed with FA/Pul nanogels. Fluorescence emission detected in both FITC and RITC channels gradually decreased as the concentration of free folic acid increased. On treating 0.5 mg/mL folic acid, almost all nanogels could not enter into KB cells. 4. Discussion Minimal procedure to obtain a polymeric nano-carrier for active drug delivery may have great impact to the pharmaceutical field, in the view of saving cost and time. In addition, the simple preparation method can provide more clearly understanding about the physicochemical property of nano-carriers and more accurately prediction about their in vivo fate as well as therapeutic effect. Recent studies on the nano-sized drug delivery carriers, however, have developed more complicated delivery systems, which must be due to the effort to prepare more efficient drug carriers. Complexity in the carrier design can improve drug loading or targeting efficiency, but multiple steps in the chemistry inevitably increase the number of unpredictable variables after all. Moreover, it will take much time and cost to utilize such drug carriers in practical fields. In those reasons, here, we suggested a simplest method to prepare nano-carriers with targeting moiety for active anti-cancer drug delivery. The initial objectives were whether amphiphilic polymers prepared by one-step chemistry could form a spontaneous nanoparticle in an aqueous solution and whether the nanoparticles could specifically interact with target cells. As a result, those two hypotheses were successfully proved by the present study. The self-organized nanogels (FA/Pul) could be generated out of just two components, water-soluble polysaccharide (pullulan) and folic acid, which was verified by DLS, CAC measurement and TEM images. Competition study using free FA and FA/Pul revealed the specific binding of nanoparticles to FA receptor-overexpressing cells. The CACs of FA/Pul conjugates were lowered by increasing the DS of FA because the FA hydrophobic enough to form self-organized nanogels. CACs of each polymer were lower than the typical critical micelle concentration (CMC) for lowmolecular-weight surfactants, such as sodium dodecyl sulfate and deoxycholic acid in water [3,13]. Such low CAC values of the FA/Pul conjugates may comprise one of the important characteristics of the polymeric amphiphiles examined in this study; i.e., a small amount of the conjugate self-organizes and maintains stability under dilute conditions. Particle stability in water appeared to be different depending on DS of FA. As displayed in TEM images (Fig. 3), FA4/Pul with low DS could not produce a complete form of nanoparticle, which shows much of small aggregates all around. In contrast, FA6/Pul and FA8/Pul showed a clear shape of nanogel. In aqueous environment, small polymer

aggregates appearing in Fig. 3a were dispersed over the solution and they accelerated inter-particular aggregation. Big aggregates around 1000 nm in Fig. 2, which gradually disappeared as DS increased are possibly explained by this phenomenon. It is well known that the DS of hydrophobic moieties has influence on the particle size as well as on the particle stability [14–17]. Drug-loading efficiency and release profile also can be controlled by the DS. FA/Pul with high DS could carry more DOX and liberate it more slowly as shown in Fig. 4. The FA4/Pul loaded only about 55% DOX in comparison with the FA8/Pul. Moreover, the half-maximal release amount of DOX from the FA4/Pul nanogel was more than twice of that from FA8/Pul. Since hydrophobic drugs are likely to be trapped in the hydrophobic cores inside nanogels, it is reasonable that particles with higher DS can hold more drugs and that liberate them more slowly [2]. Additional possibility, although it was not confirmed in this study, is that the ester linkage between FA and Pul backbone chain is likely to be cleaved in aqueous solution. Such degradation can be accelerated by low or high pH as well as enzymes such as esterase existing in lysosomal compartment of cells. Fundamentals in the design of FA/Pul included the hypothesis of ester breakage because tumor tissues have generally lower extracellular pH than normal tissues possess [18]. As addressed above, low pH around tumor might mediate the hydrolysis of ester linkages so that a burst effect of DOX could be expected. This issue, pH-dependent drug release, is not a focus in this report and requires further study to clarify the exact mechanism. Cell viability in the presence of DOX-loaded nanoparticle should be also considered in the extension of DOX release profile. Because treatment of PBS or DOX-free FA/Puls did not induce cytotoxicity of KB cells, it could be concluded that the drug carriers have good cytocompatibility. On the other hand, DOX-containing nanogels effectively lowered cellular viability, at least after 24 h. Different release rate of DOX from each nanogel may explain the viability in 12 h after treatment. Because FA4/Pul nanogel liberated DOX faster than other two nanogels, KB cells treated by FA4/Pul containing DOX showed statistically significant loss of viability. Although in vivo therapeutic activity of FA/Pul nanogels must be evaluated further, it is an important finding that the drug loading and release as well as the anti-cancer activity could be modulated simply by the DS of FA. Furthermore, specific interaction of the FA/Pul nanogels with the folate receptors (Figs. 6 and 7) promises that the nanogels can be a good candidate of an anti-cancer drug carrier with high therapeutic efficacy by an active targeting strategy. 5. Conclusion The objectives of the current study were to develop a simple fabrication method of self-organized nanogels holding both anti-cancer drug and targeting moiety. One-step chemistry successfully produced FA/Pul polymers and they spontaneously formed self-organized nanogels in an aqueous environment. Particle size did not depend on the DS of FA but stability of particle size did. In addition, loading and releasing an anti-cancer drug, DOX, could be controlled by the DS of FA. Although high DS

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showed higher loading efficiency and slower release profile, it did not affect the interaction with cells nor the cellular uptake. A competition study supported the particle specificity, which suggested a strong potential of FA/Pul nanogels as a good drug carrier for active targeting chemotherapy.

[8]

[9]

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