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Journal of Colloid and Interface Science 258 (2003) 415423 www.elsevier.


Cross-linked polyvinylpyrrolidone nanoparticles: a potential carrier for hydrophilic drugs

Dhruba Jyoti Bharali, Sanjeeb Kumar Sahoo,1 Subho Mozumdar, and Amarnath Maitra
Department of Chemistry, University of Delhi, Delhi 110007, India Received 14 December 2001; accepted 28 October 2002

Abstract Injectable hydrogel polymeric nanoparticles of polyvinylpyrrolidone cross-linked with N, N -methylene bis-acrylamide and encapsulating water-soluble macromolecules such as FITCdextran (FITCDx) have been prepared in the aqueous cores of reverse micellar droplets. These particles are 100 nm and below in diameter with a narrow size distribution. When dispersed in aqueous buffer these particles appear to be transparent and give an optically clear solution. Lyophilized powder of these nanoparticles is redispersable in aqueous buffer without any change in the size and morphology of the particles. The efciency of FITCDx entrapment by these nanoparticles is high (> 70%) and depends on the amount of cross-linking agent present in the polymeric material. The release of the entrapped molecules from these nanoparticles depends on the degree of cross-linking of the polymer, particle size, pH of the medium, and extent of loading, as well as temperature. 2003 Elsevier Science (USA). All rights reserved.
Keywords: Hydrophilic nanoparticles; PVP; FITCdextran; AOT; Reverse micelles; Drug encapsulation

1. Introduction During the past two decades there has been a growing interest in the use of hydrogel polymers as carriers for targeting drugs at specic sites in the body [15]. These polymeric materials are usually biocompatible, biodegradable, and nonantigenic compounds and are easily dispersible in water. Water-soluble drugs can be incorporated into these hydrogels. The use of hydrophilic polymeric nanoparticles as drug carriers for the purpose of drug targeting is a challenging area of research, as water-soluble drugs, including proteins and nucleic acids in the naked forms or in the forms of prodrugs, are sometimes degradable in the body system and therefore need complete protection from hostile enzymes until they can reach the targeted tissues. A considerable amount of effort has been devoted to the creation of nanoparticles for drug delivery systems acceptable for general systemic use and capable of carrying a drug to its target at the cellular or tissue level. With the advent of recombinant DNA technology, various types of proteins and
* Corresponding author.

E-mail address: (A. Maitra).

1 Present address: Department of Pharmaceutical Science, University of

Nebraska Medical Center, Omaha, NE 68198-6025, USA.

polypeptides are being used as drugs as well as vaccines. Encapsulation of these drugs into liposomes is not very effective, as the loading efciency into liposomes is very low. Hydrophobic nanoparticles cannot be used for encapsulation of these hydrophilic drugs. Most of the preparative methods being followed up till now have been emulsion-mediated processes, which produce nanoparticles of diameter larger than 100 nm with broad polydispersity [4,1012]. This size is quite large compared to the histology of the endothelial barrier, whose fenestration is around 5060 nm in diameter or in the case of vasculature in solid tumor sinusoids which are less than 100 nm in diameter [13]. Nanoparticles made of polymeric micelles are better targetable materials, particularly for solid tumors, because (i) these particles have a hydrophilic surface and (ii) their sizes are much less than diameter 100 nm [14,15]. However, the bulk cores of these particles are hydrophobic and can dissolve only hydrophobic drugs. Therefore they are not suitable for encapsulating hydrophilic water-soluble compounds. The bodys RES, mainly the Kupffer cells in the liver, usually takes up polymeric nanoparticles with hydrophobic surface and, therefore, the residence time of these particles in the blood is small. Surface coverage by amphiphilic polymeric surfactants such as poloxamers and poloxamines over the nanoparticles signicantly increases the blood circulation time but

0021-9797/03/$ see front matter 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0021-9797(02)00099-1


D.J. Bharali et al. / Journal of Colloid and Interface Science 258 (2003) 415423

does not eliminate the RES uptake [59]. As a result, the delivery of hydrophilic drugs through targeting of nanoparticles to sites other than RES presents an important challenge: to improve bioavailability. By analogy with stealth liposomes [16], this strategy requires the polymeric nanoparticles to have (i) a hydrophilic core to solubilize water-soluble drug molecules and (ii) a diameter less than 100 nm with a hydrophilic surface to evade the RES and have long residence in blood in order to be amenable for transendothelial passage. This paper describes a process of preparation of polyvinylpyrrolidone (PVP) hydrogel nanoparticles of diameter less than 100 nm using the aqueous cores of reverse micellar droplets as nanoreactors [17]. This hydrophilic polymer is known to be biocompatible and nonantigenic [18] and is therefore safe for animal experiments. Since the reverse micellar droplets are highly monodisperse and the droplet size can be varied, the nanoparticles prepared using a reverse micelle medium are nearly monodisperse with narrow size distribution dispersions. Moreover, their size can be modulated by controlling the size of the reverse micellar droplets [19]. Physicochemical characterizations of these PVP nanoparticles by size, loading ability, release behavior, and chemical stability are also reported in this paper.

2. Experimental 2.1. Materials Aerosol OT, i.e., AOT (sodium bis-2-ethylhexylsulfosuccinate) (purity > 99%), N, N, N , N -tetramethylethylenediamine (TEMED), N, N -methylene bis-acrylamide (MBA), and uorescein isothiocyanate conjugated to dextran (FITCDx) were products of Sigma, USA and were used directly without further purication. n-Hexane (99%), ammonium persulfate (APS), sodium monohydrogen phosphate, and dihydrogen phosphate, and ferrous ammonium sulfate (FAS) were procured from SRL (India) and are of high purity grade. Vinylpyrrolidone were purchased from Fluka and was freshly distilled before polymerization. Double-distilled water was used for all the work. 2.2. Preparation of cross-linked PVP nanoparticles loaded with FITC-dextran in reverse micelles AOT has been selected for reverse micelle formation because this surfactant aggregates in oil without using any cosurfactant and the reverse micellar solution can dissolve considerable amount of water and other hydrophilic materials [20]. Moreover, unlike other surfactants, the removal of AOT from the aqueous system through its precipitation as a calcium salt is easy, quantitative, and rapid. We have prepared the reverse micelles in n-hexane, as it is easily removable by evaporation from the micellar system. The reverse

micelles were prepared by dissolving AOT in n-hexane (usually 0.03 to 0.1 M of AOT solution). A typical preparative method is as follows: A quantity of 40 ml of 0.03 M AOT solution in hexane was taken and to it 280 l of freshly distilled VP, 100 l of MBA (0.049 g/ml), 20 l of TEMED, and aqueous solutions of 20 l of ammonium persulfate (20% w/v) and 50 l of FITCDx (3.2% w/v) were added. The solution was homogeneous and optically transparent. Polymerization was done at 37 C for 8 h in a thermostatted water bath with continuous stirring. After the completion of the reaction, the excess solvent (hexane) was evaporated off in a rotary evaporator and the dry mass was resuspended in 10 ml of water by sonication. Then 1-ml aliquots of 30% w/v calcium chloride solution were added drop by drop with continuous stirring to precipitate the AOT (surfactant) as calcium salt of bis-(2-ethylhexyl) sulfosuccinate, Ca(DEHSS)2 . It was then dialyzed for 2 h. Ca(DEHSS)2 was separated by centrifugation at 10,000 rpm for 10 min. The centrifuged aqueous solution contained the FITCdextran-loaded PVP nanoparticles, which were homogeneous and transparent. The precipitate of Ca(DEHSS)2 after centrifugation contained some FITCdextran-loaded PVP nanoparticles absorbed on it. The precipitated pellet was dissolved in 15 ml of hexane and the hexane solution was washed three times with 1 ml of water. The phase-separated aqueous layer was drained out and added to the previously centrifuged supernatant solution containing the PVP nanoparticles. The total aqueous dispersion of nanoparticles was lyophilized immediately to dry powder. Lyophilized nanoparticles were easily redispersible in aqueous buffer. The ow diagram for the preparation of the nanoparticles through reverse micelles is shown in Fig. 1. 2.3. Characterization of nanoparticles 2.3.1. FT-IR studies Mid-IR spectra of VP, MBA, and cross-linked PVP nanoparticles were taken in KBr pellets using a Perkin Elmer Fourier transform infrared (FT-IR) spectrophotometer (Spectrum 2000) instruments. 2.3.2. 1 H NMR studies The nuclear magnetic resonance spectra of the monomer VP and the cross-linked polymeric nanoparticles was taken using CDCl3 as solvent and in a Bruker Spectrospin Avance 300 spectrometer. 2.3.3. Size determination QELS measurements. Dynamic laser light-scattering measurements for determining the size of the nanoparticles were performed using a Brookhaven 8000 instrument with a BI200SM goniometer. An air-cooled argon ion laser was operated at 488 nm as a light source. The time dependence of the intensity autocorrelation function of the scattered intensity was derived by using a 128-channel digital correlator. The solutions used for dynamic light scattering

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Fig. 1. Flow diagram for the preparation of PVP nanoparticles through reverse micelles.

experiments were repeatedly ltered through a Millipore lter of pore size 0.2 m to remove any dust impurities. All measurements were made at 25 C. The size of the nanoparticles was determined from the diffusion of the particles using the StokesEinstein equation and a representative size distribution spectrum was plotted. TEM pictures. About 10 mg of lyophilized powder was dispersed in 50 ml buffer to have a clear solution and the samples for TEM were prepared with this. The prepared sample solution was put on a Formvar-coated grid (a 1% solution of Formvar was prepared in spectroscopic grade chloroform). A clean glass slide was dipped in Formvar solution to make a Formvar lm on the stars, the glass slide was scratched on the edges, the Formvar lm was oated on distilled water on a spherical container, and the 2090 mesh copper grids were placed upside down on the oating plastic lm. Next a piece of water lter paper was blotted on the plastic lm and lifted out from the distilled water. In this way the plastic-coated grids were prepared. On this grid, a drop of the sample solution (containing dispersed nanoparticles) was put and was allowed to air-dry. A TEM picture was taken in a JEOL JEM2000 Ex 200 Model electron microscope. 2.4. Determination of loading in the nanoparticles Total amount of lyophilized nanoparticles prepared through reverse micelles according to the procedure de-

scribed in Section 2.2 was dispersed in 10 ml of water. An aliquot of 500 l of this solution was ltered through a Millipore UFP2THK24 (100-kD cutoff) membrane lter. The free FITCDx (mol.wt. 19.3) would pass through the lter while the nanoparticles remained unltered. The amount of FITCDx present in the ltrate was determined spectrophotometrically using a UVIKON 930 spectrophotometer at a wavelength of 494 nm. The entrapment efciency (E %) was calculated from the total concentration of the added amount of FITCDx present in the system ([FITCDx]0) and that in the ltrate ([FITCDx]f) using the equation E % = [FITCDx]0 [FITCDx]f [FITCDx]0 100.

2.5. Separation of nanoparticles by gel permeation chromatography In order to separate free FITCDx from the nanoparticles, the sample was passed through a Sephacryl S-200 column (1 50 cm) preequilibrated with 50 mM PBS at a ow rate of 8 ml/h at 25 C. All the fractions collected were analyzed for their uorescence values at excitation and emission wavelengths of 495 and 520 nm, respectively, using a Shimadzu spectrouorimeter. The uorescence intensity was plotted against the fraction number. 2.6. In vitro release kinetic studies A known amount of lyophilized PVP nanoparticles encapsulating FITCDx was suspended in 10 ml buffer of de-


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sired pH in which release kinetics are to be studied. The solution was distributed in 20 micro tubes, 500 l each, and was kept at a constant temperature of 25 C. At predetermined intervals of time the solutions were ltered through a Millipore lter as indicated above to separate free FITC Dx from the loaded nanoparticles. The concentration of free FITCDx was determined spectrophotometrically by adding 100 l of ltrate in 3 ml of buffer solution (pH 8.0) and measuring the absorbance at a wavelength of 494 nm. The percentage release was determined from the equation % Release = [FITCDx]f,t [FITC Dx]0 100,

where ([FITCDx]f,t ) is the concentration of FITCDx in the ltrate at time t . The release kinetics were studied at pH 5.0, 7.0, 8.0, and 9.0 at 25 C. The release kinetics of FITCDx from nanoparticles having different degree of cross-linking was also studied in a similar way at pH 7.0. A comparative study of release behavior of this marker molecule from these PVP nanoparticles was also done in serum and aqueous buffer (pH 7.4).

3. Results and discussion 3.1. Infrared studies of the polymeric nanoparticles Cross-linked polyvinylpyrrolidone (PVP) was prepared by the vinyl polymerization of monomers (VP); ammonium persulfate (APS) was used as initiator. FTIR spectra of the monomer vinylpyrrolidone, cross-linking agent MBA, and cross-linked PVP nanoparticles are shown in Fig. 2. As shown in the gure, strong peak at 850 cm1 corresponding to the =CH2 wagging of the vinyl double bond disappeared in the spectrum of the polymer and the peaks at 1630 and 1426 cm1 for C=C and vinyl scissoring vibration disappeared in the spectrum of the polymer, indicating that the polymerization reaction had taken place. The CH stretching vibration of the polymer backbone is manifested through a strong peak at 2928 cm1 . Peaks at 16401720 cm1 correspond to >C=O stretching from pyrrolidone units. 3.2. Proton NMR spectral studies of the polymeric nanoparticles To further ensure whether the polymerization has taken place or not, a comparative study of proton NMR spectra of the monomer (VP), cross-linking agent (MBA), and polymer (PVP) was done. Figure 3 exhibits the 1 H NMR spectra of VP, MBA, and cross-linked PVP polymeric nanoparticles. The polymerization is indicated by the absence of a double doublet for two protons at = 4.4 for CH=CH2 in N -vinylpyrrolidone, due to coupling with the neighboring proton. The polymerization is also conrmed by the absence of downeld protons in the other starting compounds MBA in the range = 6.35.7 for unsaturation.
Fig. 2. FTIR spectra of (a) N -vinylpyrrolidone, (b) N, N -methylene bisacrylamide, and (c) cross-linked PVP nanoparticles.

3.3. Dependence of some physicochemical parameters on the yield of nanoparticles formation 3.3.1. The effect of initiator concentration The effect of the concentration of initiator on the yield of lyophilized PVP nanoparticles is shown in Fig. 4a. Longchain polymer cross-linked with MBA forms stable nanoparticles and therefore the monomers and small oligomers are removed from the nal nanoparticles by extensive dialysis through a 12-kD cutoff dialysis bag. Each time we took 40 ml of 0.03 M AOT in hexane as a reaction medium; to which a xed amount of 1050 l of 20% w/v APS was

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(c) Fig. 4. Effect of (a) initiator concentration, (b) temperature, and (c) monomer concentration on the yield of the PVP nanoparticles per 40 ml of 0.03 M AOT/hexane reverse micellar set.

monomers present in the aqueous core of the reverse micellar droplets, and under such circumstances the longest possible polymer chains are formed.
Fig. 3. 1 H NMR spectra of (a) N -vinylpyrrolidone, (b) N, N -methylene bis-acrylamide, and (c) cross-linked PVP nanoparticles.

added and the corresponding yield was recorded. It is clear from the gure that initially with the increase in the concentration of APS, the yield of PVP nanoparticles is increased and reached a maximum value of about 25% when 20 l of APS in added. Further increase of APS concentration led to decrease in the overall yield of the PVP nanoparticles formed. This is quite obvious from the fact that at lower APS concentrations the polymerization reaction was incomplete and, therefore, a large number of unreacted monomers and small oligomers passed through the 12-kD dialysis bag. Similarly, using higher concentration of APS also led to the formation of small oligomers that were lost during dialysis. When we calculated the number of reverse micellar droplets present in 40 ml of 0.03 M AOT in hexane solution from Ref. [21] as well as the number of perdisulfate ions added, we surprisingly saw that the maximum yield occurred when the droplets-to-APS ratio was 1 : 1. This implies that one APS molecule is enough to initiate polymerization of

3.3.2. Effect of monomer concentration A plot of the percentage of yield of the PVP nanoparticles versus the concentration of the monomer (VP) taken is shown in Fig. 4c. With the increase in the concentration of the monomer, the percentage of yield increased till 280 mg of the monomer was added and after this concentration, the yield of the PVP nanoparticles remained more or less constant. So at an optimum concentration of APS (20 l), the amount of polymer formed is increased with increased amounts of added VP and reaches a maximum value at 280 l of VP, beyond which no further yield of polymer takes place. 3.3.3. Effect of temperature The temperature is known to have a strong inuence on the rate of the polymerization reaction and the consequent yield. Usually vinyl polymerization reactions are best done at 6080 C. But with the increase in temperature, the number of active sites increases, resulting in the formation of innumerable polymeric chains of smaller length. The yield of PVP nanoparticles formed at different temperatures is shown in Fig. 4b. At lower temperatures as well as higher


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Fig. 5. A representative QELS spectrum of PVP nanoparticles.

temperatures the yields are poor. The yield is maximal at 37 C. The formation of small oligomers at higher temperatures, which are not retained by the dialysis bag, may be responsible for the lower yield. 3.4. Size and morphology of the PVP nanoparticles

The sizes and morphology of PVP nanoparticles entrapping FITCDx were measured by QELS and by TEM. The nanoparticles (0.6% cross-linking) encapsulating FITCDx were found to be around 50 nm diameter as measured by QELS (Fig. 5). The same particles exhibit diameter of about 3035 nm in the TEM pictures (Fig. 6a). We believe that hydration and swelling of the particles in aqueous buffer or in the aqueous cores of reverse micellar droplets may be the reason for observing larger size by QELS measurements. The mean diameters of the nanoparticles have been found to be below 100 nm in all cases, depending on the size of the aqueous cores of reverse micellar droplets in which these particles have been prepared. Surprisingly, the size of the nanoparticles is always larger than that of the aqueous cores of the host reverse micellar droplets as measured by QELS. In our earlier communication [19] we explained this as due to the dynamic behavior of the reverse micellar droplets. These droplets form transient clusters, leading to the intermixing of the aqueous hydrophilic materials among different droplets. As a consequence, the polymer chains of various droplets interconnect among themselves, forming larger nanoparticles. The particle size, as shown in Fig. 7, remains more or less constant at about 50 nm diameter till the host droplet size is about 5 nm. A sharp rise in particle size takes

Fig. 6. TEM pictures of PVP nanoparticles: cross-linking agent (a) 0.6%, (b) 1.2%.

place when the droplet size is above 5 nm in diameter. When the size of the micellar droplets is larger than 10 nm, the particles formed are too big to remain dispersed in the micellar system. These large particles settle down at the bottom of the liquid and their size cannot be measured by the QELS experiments. The quasi-sudden increase in particle size at and beyond a certain droplet size has the same trend as observed in the case of polyacrylamide nanoparticles [19]. The size of the nanoparticles also depends on the amount of the cross-linking agent, MBA, present in the polymer composition. The nanoparticle sizes seen in Figs. 6a and 6b increased from 35 to 80 nm diameter when the amount of MBA was increased from 0.6% w/w to 1.2% w/w of the polymeric material. With the increased concentration of the cross-linking agent, the probability of connection of interdroplet polymer chains increases. This facilitates the interlinking of the primary polymeric particle from one droplet with the polymer chain of another droplet during the lifetime of transient droplet dimers, resulting in the formation of larger particles.

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Fig. 7. Dependence of FITCDx-loaded nanoparticle size on the size of the reverse micellar droplets in which these nanoparticles were prepared. Loading of FITCDx = 3.2% w/w of the polymeric material; MBA = 1.2% w/w of polymeric material.

Fig. 8. Separation of PVP nanoparticles from free FITCDx by gel permeation chromatography.

3.5. Separation of the loaded nanoparticles Figure 8 shows the gel permeation chromatrogram for FITCDx-loaded PVP nanoparticles just after equilibrium in PBS buffer at pH 8.0. The detection of FITCDx was carried out spectrophotometrically by measuring its emission at 520 nm. PVP nanoparticles were eluted at the gel exclusion volume, consistent with the apparent molecular weight of nanoparticles, which is probably greater than 106 g/mol [24]. FITCDx, however, comes out at a higher elution volume. The entrapment and retention of FITCDx in the PVP nanoparticles may be a result of the highly crosslinked solid like polymeric core of the particles, from which the release of FITCDx is constrained due to its high molecular weight. From the gel permeation chromatography, the approximate molecular weight of the polymeric nanoparticles was found to be about 3500 kD. 3.6. Entrapment efciency The entrapment efciency (E %) of these PVP nanoparticles entrapping FITCDx was found to be about 70% when 2.5% w/w of FITCDx was added to the polymeric material. The E % was, however, found to be more or less constant even at a higher dissolution of FITCDx (up to 3.5% w/w), above which E % falls rapidly. The E %, however, remains practically constant at about 70% within the broad range of PVP nanoparticle size from 25 to 95 nm diameter. On the other hand, E % for FITCDx can be slightly increased by increasing the concentration of cross-linking agent. 3.7. In vitro release kinetic studies in aqueous buffer and in blood serum In order to understand the stability of FITCDx-entrapped PVP nanoparticles dispersed in aqueous buffer, we have measured the release of FITCDx from these nanoparticles under different physicochemical conditions. The cumulative percentage of FITCDx released from nanoparticles containing different amounts of cross-linking agent at different

time intervals has been shown in Fig. 9a. The in vitro release from the various formulations never reached 100%. The duration of in vitro release of entrapped FITCDx was prolonged with increased amount of cross-linking agent (CLA) (e.g., 1.2% CLA: 34 weeks). The effect is probably due to the formation of densely cross-linked matrix [22,23], which affect the pore size and retard the solvation kinetics of the polymer. Increasing the amount of cross-linking agent can also increase the size of the particle [19], and consequently can retard the release rate also. FITCDx is a large molecule (mol.wt. 19.3 kD). Therefore, simple diffusional release of the molecule from the polymer matrix of the nanoparticles is difcult unless the polymer is swelled or eroded in an aqueous medium. PVP hydrogel cross-linked with MBA has a structure in which polymeric erosion through cleavage may take place only through the hydrolysis of the amide bond of the cross-linking agent with consequent generation of a loose polymeric network [24]. However, Torchilin et al. [25] indicated that the rate of cleavage of amide bonds in MBA cross-linked polymer is extremely low, particularly when the networks are prepared with more than 1% w/w MBA. Therefore, it is certain that the release of FITCDx from the PVP nanoparticles precedes polymer degradation. The plausible route of drug release, under these circumstances, is therefore believed to be due to a diffusion-controlled process from the highly swollen gel nanoparticles. To understand this we have determined the prole of FITCDx release from the PVP nanoparticles at different pH. PVP is a hydrophilic polymer, which swells in water ([18, Chap. 1, pp. 117] and [2527]). The characteristics of in vitro release of entrapped FITCDx from PVP nanoparticles at pH 5.0, 7.0, 8.0, and 9.0 were determined at 25 C and the results are shown in Fig. 9b. It is evident from the gure that the release rate is slower both in higher and in lower pH and it attains a maximum at pH around 7.0. While in two weeks, time about 43% of FITCDx is released in pH 5.0 solution, this is increased to about 70% during the same period in pH 7.0. This behavior clearly indicates that PVP hydrogel nanoparticles may undergo swelling in aqueous dispersion and the swelling is maximal in neutral pH.


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Fig. 9. (a) Release kinetics of FITCDx from PVP nanoparticles in aqueous buffer (7.0). The amount of cross-linking agent (MBA) is shown in each curve. FITCDx loading = 3.2% w/w of polymeric material. (b) Release kinetics of FITCDx from PVP nanoparticles (1.2% w/w MBA) dispersed in aqueous buffers of different pH (indicated against each curve). FITCDx loading = 3.2% w/w of polymeric material.

actors has been optimized. The technology has enabled us to prepare hydrogel nanoparticles of diameter below 100 nm encapsulating water-soluble materials. The particles can be lyophilized and redissolved in aqueous buffer without having any change of their size and surface morphology. These particles are stable in aqueous dispersion and increasing the cross-linkage can modulate the release of the encapsulated compound.

Fig. 10. Release kinetics of FITCDx from PVP nanoparticles (1.2% w/w MBA) dispersed in aqueous buffer (pH 7.0) containing 3.2 and 6.4% w/w FITCDx in total polymeric material. [1] J.E. Tomlin, S.S. Davis (Eds.), Site Specic Drug Delivery, Wiley, Chichester, 1986. [2] S.S. Davis, L. Illum, Biomaterials 9 (1988) 111115. [3] S.S. Davis, L. Illum, S.M. Mognimi, M.C. Davies, C.J.H. Porter, I.S. Muir, A. Brindley, N.M. Christy, M.E. Norman, P. Williums, S.E. Dunn, J. Controlled Release 24 (1993) 157163. [4] E. Allerman, R. Gurny, E. Doelken, Eur. J. Pharm. Biopharm. 39 (1993) 173191. [5] S. Stolnik, L. Illum, S.S. Davis, Adv. Drug Delivery Rev. 16 (1995) 195214. [6] V.P. Torchilin, V.S. Trubetskoy, Adv. Drug Delivery Rev. 16 (1995) 141145. [7] R. Gref, A. Domb, P. Quelle, T. Blunk, R.H. Muller, J.M. Verbatz, R. Langer, Adv. Drug Delivery Rev. 16 (1995) 215233. [8] J.S. Tan, D.E. Buttereld, C.L. Voychek, K.D. Caldwell, J.T. Li, Biomaterials 14 (1993) 823833. [9] C.J.H. Porter, S.M. Mognimi, L. Illum, S.S. Davis, FEBS Lett. 305 (1992) 6266. [10] S.S. Davis, L. Illum, in: G. Gregoriadis, G. Poste (Eds.), Targeting of Drugs, Anatomical and Physiological Considerations, Plenum Press, New York, 1988, pp. 177187. [11] T. Curt, in: M. Rosott (Ed.), Controlled Release of Drugs: Polymers and Aggregated Systems, VCH, New York, 1989. [12] S.S. Davis, TIBTECH 15 (1997) 217224. [13] H.S. Mayerson, C.G. Wolfram, H.H. Shirby, K. Wasserman, Am. J. Physiol. 198 (1959) 155160. [14] K. Kataoka, G.S. Kwon, M. Yokoyama, T. Okano, Y. Sakurai, J. Controlled Release 24 (1993) 119132. [15] G.S. Kwon, T. Okano, Adv. Drug Delivery Rev. 21 (1996) 107116. [16] N.M. Wu, D. Da, T.L. Rudoll, D. Needham, A.R. Whorton, M.W. Dehirst, Cancer Res. 53 (1993) 37653770. [17] A.N. Maitra, P.K. Ghosh, K. De Tapas, S.K. Sahoo, US Patent 5,874,111, 1999.

The in vitro release of FITCDx from PVP nanoparticles is also dependent on the loading of the dye. Figure 10 demonstrates that in the case of 3.2% w/w loading of FITC Dx in PVP polymer cross-linked with 0.3% MBA, nearly 95% release of drug takes place in 1012 days. The release is only about 60% from the nanoparticles during the same period when the dye loading is 6.4% w/w in the same polymeric material. A larger population of dye (6.4%) within the same volume of nanoparticles may lead, at least partly, to the association of dye molecules as dimers, trimers, etc. or even to the formation of solids clusters [28] inside the matrices of the nanoparticles. At low loadings of the compound, it remains in the form of a molecular dispersion inside PVP nanoparticles. When the concentration of dye inside the core of the particle is very high, a part of it is associated or clustered, which has to be dissolved and released more slowly out of the nanoparticles.

4. Conclusions A novel technique of preparing hydrogel polymeric PVP nanoparticles with well-regulated size and size distribution using aqueous cores of reverse micellar droplets as nanore-

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