Doi:10.1016/j.jconrel.2003.10.012

Journal of Controlled Release 94 (2004) 323 – 335 Incorporation and release behavior of hydrophobic drug in functionalized poly(D,L-lactide)-block–poly(ethylene oxide) Jaeyoung Lee, Eun Chul Cho, Kilwon Cho* Department of Chemical Engineering, School of Environmental Engineering, Pohang University of Science and Technology, 790-784 Pohang, South Korea Received 22 May 2003; accepted 9 October 2003 The poly(ethylene oxide) – poly(lactide) (PEO – PLA) block copolymers containing a small quantity of carboxylic acid in the PLA block were synthesized. The microscopic characteristics of nanoparticles with carboxylic acid content in the copolymerwere analyzed, and the effect of specific interactions between the copolymer and the model drug on the drug loading capacityand the release behavior were investigated systematically. The sizes of nanoparticles prepared by a dialysis method are withinthe range of 30 – 40 nm. The nanoparticles prepared from functionalized block copolymers have a very low critical micelleconcentration (CMC) value as low as f10
3 mg/ml, which indicates a good stability of the nanoparticles in spite of thepresence of carboxylic acid. The drug loading efficiency of nanoparticles dramatically increased when carboxylic acid contentwas increased in the block copolymer. This result may be attributed to the increase of interactions between the copolymer andthe drug. The release rate of the drug was much slower from nanoparticles containing higher amounts of carboxylic acid in thecopolymer, which might be associated with the enhanced interaction between the carboxylic group of copolymers and the drug.
These experimental results suggest that the nanoparticles prepared from functionalized PEO – PLA block copolymers could be agood candidate for an injectable drug delivery carrier.
D 2003 Elsevier B.V. All rights reserved.
Keywords: Block copolymer; Micelle; Nanoparticle; Drug delivery; Careboxylic acid block copolymer has been the focus of researchespecially because of the nano-character in self-as- Over the past few decades, biodegradable nano- sembling systems This type of diblock copoly- particles have attracted considerable interest as an mer can form a spherical micelle structure in an effective drug carrier device. Various polymers have aqueous media. The hydrophobic blocks of the co- been used in drug delivery systems. The amphiphilic polymer form the core of the micelle, while thehydrophilic blocks form the corona or outer shell.
The hydrophobic micelle core serves as a microenvi-ronment for incorporating hydrophobic drugs such as * Corresponding author.
E-mail address: [email protected] (K. Cho).
anti-cancer drugs while the outer shell serves as 0168-3659/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2003.10.012 J. Lee et al. / Journal of Controlled Release 94 (2004) 323–335 a stabilizing interface between the hydrophobic drug a hydrophobic drug is needed with the functional and the external medium. Since most drugs have a group of core block.
hydrophobic character, the drugs can be easily incor- To solve this problem and increase the drug load- porated into the micelle by simple physical entrap- ing capacity compared to unfunctionalized poly(eth- ment through dialysis or by an oil/water emulsion ylene oxide) – poly(lactide) (PEO – PLA) block method. The solubility of hydrophobic drug in the copolymer, we synthesized the PEO – PLA diblock aqueous media is greatly increased by the use of copolymers containing a small quantity of carboxylic micelle. Thus, incorporating a drug in the micelle is acid in the PLA block and prepared functionalized an effective method of preparing an efficient drug nanoparticles. This study analyzed the microscopic delivery system. Biodegradable and biocompatible characteristics of nanoparticles with carboxylic acid polymers such as poly(lactide) (PLA) poly(q- content in the copolymer and investigated the effect of caprolactone) (PCL) poly(h-benzyl L-aspar- specific interaction between the copolymer and the tate) (PLBA) and poly(g-benzyl L-glutamate) model drug on the drug loading capacity and the (PLBG) have been used mostly for the core release behavior systematically.
material of micelle. Moreover, poly(ethylene oxide)(PEO) is a non-toxic, highly hydrated polymer thatstabilizes the surfaces in aqueous systems, and it is effective in preventing the adsorption of proteins andadhesion of cells. Therefore, PEO has been used as the outer shell material of micelle for a long-circulat-ing drug carrier.
Methoxy terminated poly(ethylene oxide) (MePEO) Studies on the polymeric micelles composed by with molecular weight of 5000 was purchased from the PEO as hydrophilic block and PCL, PLA, PLBA, Fluka. LAC was supplied by Aldrich and recrystal- PLBG as hydrophobic block have been carried out lized from ethyl acetate. Stannous 2-ethylhexanoate by many groups The size, stability, drug was provided by Sigma Chemical and purified by loading capacity, release kinetics, circulation time and biodistribution of micelles are several key prop- quinoline) hydrochloride was obtained from Sigma erties that have been studied. These properties are Chemical, and a papaverine free base (PAP) was affected by the molecular weight, the composition, prepared by increasing the pH of solution above 10.
and the chemical structure of diblock copolymer.
L-Aspartic acid and bromoacetylchloride were pur- Therefore, these studies focused on correlating the chased from Aldrich and Fluka, respectively. All structure of block copolymer and the properties of other chemicals used were of reagent grade and micelle. The block copolymers developed so far used without further purification.
either contain no functional groups at all in thehydrophobic block like as PCL, PLA or contain 2.2. Snythesis of functionalized monomer and diblock excessive functional groups like as poly(aspartic acid) When the functional groups are notcontained in the hydrophobic block of the copoly- mer, no special interaction such as hydrogen bonding 2,5-dione (BMD) (5) was prepared from L-aspartic can be expected between the copolymer and the acid according to the following procedures drug. Consequently, drug is not very well loaded The synthesis of PEO – PLA diblock copolymer into the micelle. And this copolymer has a relatively containing carboxylic group (7) was also conducted low drug loading efficiency. In the case of copoly- according to the following procedures mers containing excessive functional groups, the PEO – PLA diblock copolymers containing benzyl copolymers may be water-soluble and unable to form moiety (6) were synthesized by ring-opening copoly- the micelle in an aqueous medium. Thus, attaining merization of LAC and BMD in the presence of the amphiphilicity of the copolymer is encountered MePEO homopolymer with stannous 2-ethylhexa- with a problem—that is, the chemical conjugation of noate as a catalyst. The weighed amounts of MePEO, J. Lee et al. / Journal of Controlled Release 94 (2004) 323–335 Scheme 1. Synthetic scheme of functionalized monomer (BMD).
LAC, BMD, and stannous 2-ethylhexanoate were dispersed to this solution, the flask was evacuated, placed in a 100 ml round flask equipped with a connected to a hydrogen depot and filled with hydro- vacuum cock. The flask, filled with nitrogen gas and gen gas. The mixture was then stirred vigorously with evacuated, was heated up to 160 jC, and the mixture a magnetic stirrer and reacted with hydrogen for 15 h.
was stirred for 2 h. While the mixture was being stirred After the theoretical amount of hydrogen had been under these conditions, the viscosity of the product absorbed, the mixture was filtered to remove the increased gradually. The product was dissolved in 50 catalyst. The filtrate was condensed under reduced ml of chloroform and precipitated in an excess amount pressure and precipitated in a large excess of diethyl of diethyl ether, which was filtered and dried in a ether. The final product was filtered and dried in a vacuum oven.
PEO – PLA diblock copolymer containing carbox- ylic group (7) was obtained by the catalytic debenzy- 2.3. Preparation of nanoparticles lation of its precursor (6) in the presence of hydrogengas. A solution of 4 g of (6) in 125 ml of ethanol/ The nanoparticles (micelles) containing a hydro- dioxane (25:75) was poured into a flask of 500 ml.
phobic drug were prepared according to the dialysis After 1 g of charcoal coated with palladium was method Diblock copolymer (200 mg) was dis- Scheme 2. Synthetic scheme of PEO – PLA block copolymer containing carboxylic acid.
J. Lee et al. / Journal of Controlled Release 94 (2004) 323–335 solved in 10 ml of DMF followed by adding 150 mg determined by a dynamic light scattering (DLS) of drug (PAP) and stirred at room temperature. This instrument (Brookhaven BI-9000AT) using an argon solution was filtered to remove dust particles and ion laser at 25 jC. The intensity of the scattered light dialyzed for 24 h against 2 l of ultra-pure water using was detected with a photomultiplier as a function of cellulose dialysis membrane (Mw cut off: 6000 – the scattering angle. The signal from the photomulti- 8000). The water was exchanged to fresh water once plier was digitized via an amplifier-discriminator and every 5 h. The nanoparticle solution was filtered to was fed into a correlator. Intensity autocorrelation eliminate the unloaded drug and aggregated particles.
function was fitted by using the second cumulant The pore size of Teflon filter was 0.45 Am. A part of method. The samples for DLS measurement were nanoparticle solution was frozen and lyophilized by a prepared by diluting the nanoparticles solution. And freeze dryer system to obtain dried nanoparticles and then, the solution was filtered through a 0.45 Am also to calculate the concentration of nanoparticles Teflon filter. The concentration of solution was about solution prepared in this process. The remaining part 0.1 wt.%. This concentration is sufficiently diluted so of the nanoparticles solution was frozen to prevent that the multiple scattering due to high concentration degradation of diblock copolymer. The nanoparticles of nanoparticles may not occur.
that are not containing a drug were also preparedfollowing the same method as above.
2.5. Fluorescence measurements 2.4. Characterizations In order to determine the critical micelle concentra- tion (CMC) of diblock copolymeric nanoparticles in The chemical structure of synthesized monomer, distilled water, fluorescence measurements were car- and the composition and the molecular weight of ried out using pyrene as probe Pyrene pre- the copolymers were determined by using a proton dissolved in acetone was added to the test tube, and nuclear magnetic resonance (NMR) instrument the solvent was evaporated. Different amounts of (Bruker DRX500). The molecular weight and its nanoparticles solution and distilled water were added distribution of diblock copolymer were characterized to this tube and made a different concentration of by using gel permeation chromatography (GPC) diblock copolymer ranging from 10
6 to 10
1 mg/ (Waters 600E) calibrated using the standard poly- ml. The concentration of pyrene used was 6.0  10
7 styrene at room temperature. Tetrahydrofuran was M. The solution was incubated at room temperature used as an eluent at a flow rate of 0.8 ml/min. The with mild stirring for pyrene to equilibrate between the thermal properties of the PAP and drug containing nanoparticle and the aqueous phase completely. Fluo- nanoparticles were determined by a differential rescence excitation spectra were obtained as a function scanning calorimetry (DSC) instrument (Perkin – of the concentration of diblock copolymers using Elmer DSC 7) at a scanning rate of 10 jC/min.
spectrofluorometer (Shimadzu RF-5301PC). Experi- The X-ray photoelectron spectroscopy (XPS) instru- ments were conducted with emission wavelengths of ment (VG instrument) was used to investigate the 390 nm. Excitation and emission bandwidths were 3.0 surface chemistry of nanoparticles. The source was and 1.5 nm, respectively. Excitation spectra were a monochromated Al K-a X-rays.
obtained from the scanning excitation spectrum of each Aqueous dispersion of nanoparticles was examined sample from 300 to 360 nm, fixing the emission by using a transmission electron microscopy (TEM) wavelength at 390 nm. The CMC was determined by instrument (JEOL). Specimens were prepared by taking a mid-point of the copolymer concentration at dropping the nanoparticles solutions onto carbon which the relative excitation fluorescence intensity coated EM grids. The solution on the grid was ratio measured at 335 – 333 nm was varied.
refrigerated in liquid nitrogen and lyophilized by afreeze dryer. The nanoparticles on the grid were 2.6. Drug loading amount stained by 20 wt.% of phosphotungstic acid. Speci-mens were vacuum dried before examination. Average The drug containing nanoparticles were dissolved to size of nanoparticles in the aqueous solution was dichloromethane. The amount of papaverine free base J. Lee et al. / Journal of Controlled Release 94 (2004) 323–335 (PAP) entrapped was determined by measuring the UV 3.2. Synthesis of functionalized diblock copolymer absorbance at 238 or 279 nm. A calibration curve wasconstructed using different concentrations of free PAP BMD (5) has a ring-opening polymerizability in dichloromethane. The weight % of the PAP content comparable to glycolide and lactide The copo- entrapped into the core of nanoparticles was calculated lymerization of MePEO, LAC, and BMD was car- from the weight of the initial drug loaded nanoparticles ried out in bulk with stannous 2-ethylhexanoate. The and the amount of drug incorporated.
MePEO/(BMD + LAC) weight ratio in the feed isfixed at 2/3, and only the BMD/LAC weight ratio is 2.7. Drug release experiment changed from 5/95 to 15/85. When the diblockcopolymer has a large amount of carboxylic groups Nanoparticles solutions containing a known in the hydrophobic block, the diblock copolymers do amount of drug were sealed in a dialysis bag (Mw not form the nanoparticles in the aqueous solution.
cutoff: 6000 – 8000), and the drug was released into Therefore, the weight ratio of BMD in the hydro- 250 ml of phosphate buffer solution (pH 7.4, 0.01 M) phobic block is restricted up to 0.15. The NMR at 37 jC. The release medium (5 ml) was withdrawn spectrum of PEO – PLA diblock copolymer contain- at pre-determined time intervals and replaced with an ing BMD (6) shows the peak at d = 7.28 owing to equivalent volume of fresh buffer. The content of the the benzene group in the copolymer, which indicates drug released was directly determined from the ab- that BMD should be introduced to the diblock sorbance at 238 nm.
copolymer. However, the NMR spectrum of PEO –PLA containing carboxylic acid (7) does not nearlyshow the peak at d = 7.28, which means that the 3. Results and discussion pendant benzyl ester can readily be removed bycatalytic hydrogenolysis after copolymerization to 3.1. Synthesis of functionalized monomer yield a new carboxyl-functionalized PEO – PLAdiblock copolymer Also, GPC diagrams show The synthesis of BMD (5) was carried out only the single peak regardless of samples. These according to The reactions of up to (4) results confirm that PEO – PLA diblock copolymers progressed well, and the yields of products were containing carboxylic acid in the PLA block are higher than 95%. However, the yield of BMD was quite a low 20% because the reaction of the final shows the results of the characterization for step was progressed by the internal cyclization diblock copolymers. The compositions and Mw of between bromoacetyl group and carboxylic group copolymers were determined by proton NMR spec- of (4) whereas BMD with high purity could be troscopy and GPC measurement. The molecular obtained by a sublimation method because BMD weight % of PLA block in the copolymers was has the property of sublimation. The onset of melt- obtained from the peak intensity of the methylene ing peak of BMD synthesized is 150.8 jC (DSC proton (d = 3.65) of the PEG chain and the methyl thermogram of BMD is not shown here), which is proton (d = 1.58) of the PLA chain. The Mw and the quite consistent with the reference value (150 jC) weight composition of each block copolymers are The chemical structure of BMD is also ana- controlled similarly, but the content of carboxylic acid lyzed by a proton NMR method. Each hydrogen is controlled differently Thus, it was pos- atom of BMD is assigned to each peak of NMR sible to examine only the effect of the carboxylic acid spectrum. The integral area ratio of each peak in the copolymer on the physicochemical properties of corresponds accurately to the number of hydrogen the nanoparticles. The probability of forming the of each group in the chemical structure of BMD.
homo sequence of BMD is negligibly small at the Especially, the observation of the characteristic quar- present relatively low BMD/LAC ratios in the feed tet at d = 5.0 – 5.1 ppm in the spectrum was good composition. Consequently, the BMD unit sequences proof for its cyclic structure Thus, BMD with of PLA block are thought to be random The high purity was successfully synthesized.
number of carboxylic acid introduced in the PLA J. Lee et al. / Journal of Controlled Release 94 (2004) 323–335 Table 1Characteristics of functionalized PEO – PLA diblock copolymers a Mn, number-average molecular weight.
b PDI, Mw GPC/Mn GPC.
c Estimated from NMR spectrum.
d Weight % of BMD introduced in the hydrophobic block (PLA).
block was about 1 – 4, which indicated a small quan- hydrophobic block does not give a large effect on the tity of carboxylic acid.
formation of nanoparticles. Also, the sizes of nano-particles containing the drug were almost similar 3.3. Size of nanoparticles compared to the ones containing no drug. This resultmay be attributed to the low content of drug in the The size of functionalized diblock copolymeric nanoparticles was investigated by a DLS method.
The size and morphology of nanoparticles were The correlation function obtained from scattered in- further observed by a TEM technique. shows tensity was fitted by Eq. (1). The diffusion coefficient the nanoparticles stained by the phosphotungstic of nanoparticles (D) was obtained from the slope of acid. Because the phosphotungstic acid is a negative decay rate (C1) versus scattering vector ( q2) plot.
staining agent, the color of the stained region isshown as white brown on the TEM photograph y ¼ A exp½
C1t þ ðC2=2Þt 22 þ b The size and the size distribution of the nanoparticlesare clearly investigated. TEM photographs show that Also, the hydrodynamic radius of nanoparticles the morphology of nanoparticles is of spherical type, was determined by using the Stokes – Einstein Eq.
that the diameter of nanoparticles is about 20 nm, and that its distribution is quite uniform. It is alsoobserved that even in the case of containing BMD 19.5 wt.% in block copolymer the nano- particles are well formed. The diameter of nano- where, k, Boltzmann's constant; T, absolute tempera- particles observed by TEM is smaller than its ture, and g, viscosity of the solvent. Generally, micelleformation occurs as a result of two forces. One is anattractive force that leads to the association of mole- Table 2Characteristics and drug solubility of PEO – PLA nanoparticles cules while the other one is a repulsive force thatprevents unlimited growth of the micelles. Therefore, the micelle size is mainly determined by the relative magnitude and balance of these two forces. The sizes of nanoparticles obtained from DLS experiment were about 30 – 40 nm which are suitable for an injectable drug carrier. However, within the experi- mental range of carboxylic acid content, there is no special relation between the size of nanoparticles and the carboxylic acid content in the copolymer. This a Determined by a DLS experiment.
means that a small quantity of carboxylic acid in the b The concentration of PEO – PLA nanoparticles used is 1 wt.%.
J. Lee et al. / Journal of Controlled Release 94 (2004) 323–335 3.4. Critical micelle concentration The CMCs of functionalized diblock copolymers were determined by a fluorescence spectroscopymeasurement. Pyrene was chosen as a fluorescentprobe because of its photochemical properties suitablefor an effective probe Pyrene molecule had astrong hydrophobic character and a very low solubil-ity in water. Because pyrene preferentially solubilizeditself into the hydrophobic region of nanoparticles,the fluorescence intensity was greatly affected by theenvironmental change around pyrene. We can observethe shift of the peak at the excitation spectra with theincreasing concentration of block copolymer 2(a)). Specifically, the maximum peak for pyrene,which is at 333 nm in water, was shown to shift to335 nm upon addition of block copolymer. The CMCwas determined by taking a mid-point of the copol-ymer concentration at which the relative excitationfluorescence intensity ratio of I335 /I333 was variedThe CMC values of block copolymers areslightly increased with the content of carboxylic acidin the hydrophobic block This result maybe attributed to the increase in the hydrophilicity ofhydrophobic block with the increasing content ofcarboxylic acid. Because the absolute value of CMCis very low, however, the stability of nanoparticles isnot affected greatly by the content of carboxylic acidwithin this range.
The physical state of the inner core region of nanoparticles was further characterized by the NMRstudy. The freeze-dried nanoparticles were dispersedinto the D2O, which were filtered to remove theaggregates, and then measured by using 500 MHzproton NMR instrument. shows the NMR Fig. 1. TEM micrographs of PEO – PLA nanoparticles stained with spectrum of nanoparticles (BMD 19.5 wt.%) mea- phosphotungstic acid: (a) BMD 0 wt.%, (b) BMD 19.5 wt.%.
sured in the D2O solution. Due to the limited mobilityof PLA chains in the core of the nanoparticles, the diameter obtained from the DLS experiment. The intensity of proton peak (d = 1.55) originated from diameter of nanoparticles obtained from the DLS PLA was dramatically reduced compared to the one in experiment reflects the hydrodynamic diameter of the CDCl3 solution, where the formation of nano- nanoparticles that are swelled by water molecules, particles was not expected The small, whereas the diameter of nanoparticles observed by broad signals in the NMR spectrum indicate restricted TEM shows that of dried nanoparticles. Therefore, motions of these protons within the core of nano- an increase in the nanoparticles size obtained from particles. This suggests that the core of the polymeric DLS compared to that of TEM is assumed to be nanoparticles is a very rigid structure. This result also caused by the hydration of the shell portion of offers one of the evidences that functionalized PEO – PLA diblock copolymers may associate to form J. Lee et al. / Journal of Controlled Release 94 (2004) 323–335 Fig. 2. Fluorescence excitation spectra (a) of pyrene as a function of PEO – PLA (BMD 19.5 wt.%) concentration (mg/ml) in water. (b) Plot ofthe intensity ratio from pyrene excitation spectra vs. log C. [Py] = 6.0  10
7.
stabilized nanoparticles in the water. This behavior of (d = 3.65) originated from the outer corona region PEO – PLA nanoparticles was in contrast with low (PEO) of nanoparticles was very high like that of free molecular weight amphiphiles, which typically molecules. This result indicates that there is no exhibited liquidlike cores and a relatively higher difference on the mobility of the PEO chain between mobility. On the other hand, the proton signal nanoparticles and free molecules. Based on fluores- J. Lee et al. / Journal of Controlled Release 94 (2004) 323–335 Fig. 3. NMR spectra of PEO – PLA nanoparticles (BMD 19.5 wt.%) with different solvent: (a) D2O, (b) CDCl3.
cence spectroscopy and NMR measurement, it is drug were dissolved into the DMF solvent together, speculated that a small quantity of carboxylic acid in and dialyzed against water, then the precipitate was the PLA block does not have much effect on the filtered out, which was unloaded PAP. It is estimated formation and stability of nanoparticles.
that the aqueous solution was saturated with PAP, andthe PAP was loaded as a maximum content into the 3.5. Drug loading capacity nanoparticles. Therefore, the content of the drugdetermined by this method indicates the drug loading The amount of drug incorporated into PEO – PLA capacity of nanoparticles. The drug loading capacity nanoparticles was measured by UV spectrometer. The of nanoparticles was greatly increased with the con- freeze-dried nanoparticles were dissolved into CHCl3 tent of the carboxylic acid in the block copolymers solvent, and then UV spectra were obtained. The In the case of functionalized nanoparticles content of drug loaded within the nanoparticles was (BMD 19.5 wt.%), the weight % of the loaded drug calculated from the absorbance of PAP at 279 nm.
into the nanoparticles was 14.9. This value is more This wavelength was not interfered by the presence of than four times the unfunctionalized nanoparticles, block copolymer. When the nanoparticles solution which do not contain carboxylic acid. In order to was prepared, the copolymer and a large excess of explain this result, we had to consider the physical and J. Lee et al. / Journal of Controlled Release 94 (2004) 323–335 drug Because the number of carboxylic acidin block copolymer was very low, direct evidence onthe existence of hydrogen bond between polymer anddrug could not be found by spectroscopic technique.
However, the drug loading results clearly show that asmall quantity of carboxylic acid in the block copol-ymer has a profound effect on the drug loadingcapacity of nanoparticles.
The compatibility between the loaded drug and the core-forming block determines the efficiency of drugincorporation Polymer drug interaction suggeststhat the largest amount of drug loaded per micelle willbe reached when the core-forming block is mostsuitably matched with the drug to be loaded. There-fore, in order to enhance the encapsulation of thedrug, the compatibility between polymer and drugshould be increased. For example, the compatibilityincrease can be achieved by attaching a compatible Fig. 4. Drug loading capacity of PEO – PLA nanoparticles as a moiety such as fatty acid to the core-forming block function of weight % of BMD in hydrophobic block.
In our study, we utilize the hydrogen bondingbetween polymer and drug as another method to chemical state of the core of nanoparticles. Due to the improve the drug loading capacity of nanoparticles.
rigid and glassy state of the core, which was estimated So, we could anticipate that any kind of drug con- from NMR study the carboxylic acid could taining oxygen or nitrogen in their structure could be be non-ionized, and the water content could be very efficiently encapsulated in the nanoparticles contain- low in the core of nanoparticles. The hydration of ing carboxylic acid.
carboxylic acid was not sufficient to effectively block Meanwhile, when only the drug without the co- the hydrogen bonding between polymer and drug.
polymer was dialyzed in the same condition, the This led us to speculate that the increase of drug saturated concentration of drug was a very low loading in the functionalized nanoparticles was main- 0.0287 mg/ml. However, when the block copolymer ly attributed to the hydrogen bonding between the was used for incorporation of drug, the solubility of hydrogen of carboxylic acid in the copolymer and the drug in water was greatly increased, especially in the unpaired electron of nitrogen or oxygen atom in the case of functionalized diblock copolymer Fig. 5. Schematic diagram showing the interaction between PEO – PLA copolymer and drug (PAP).

J. Lee et al. / Journal of Controlled Release 94 (2004) 323–335 Therefore, in order to increase the water solubility of the core of nanoparticles as an amorphous form and hydrophobic drug, the use of PEO – PLA diblock does not nearly exist at the surface of nanoparticles.
copolymer containing a small quantity of carboxylicacid would be a very useful method.
3.6. Degradation of nanoparticles We further characterized the drug loaded nanopar- ticles by DSC and XPS. shows the DSC The degradation of nanoparticles in phosphate thermograms of drug (PAP) and drug loaded nano- buffered solution (pH 7.4, 0.01 M, 37 jC) was particles (BMD 19.5 wt.%). Although the weight % of monitored in a DLS experiment The concen- drug in the nanoparticles was 14.9, the characteristic tration of nanoparticles solution was 0.1 wt.%, which melting peak of drug (160 jC) was not present at all.
is suitable for DLS measurement. As the degradation Moreover, the XPS spectrum of nanoparticles does not of nanoparticles progressed, the correlation function show the peak of nitrogen from the included drug of scattered intensity deviated from a single expo- 6(b)). These results indicate that the drug is dispersed in nential decay. Because of the broad distribution ofparticle size, the correlation function could not befitted by Eq. (1). Thus, this point of time was takenas the time required for degradation of nanoparticles.
Although the degree of the degradation of nano-particles could not be quantified by using thismethod, it could be compared with the carboxylicacid content in the block copolymer. The timerequired for degradation of nanoparticles was de-creased with the content of carboxylic acid in theblock copolymer. In the case of functionalized nano-particles (BMD 19.5 wt.%), the time required fordegradation of nanoparticles is about 6 days. Where-as in the case of unfunctionalized nanoparticles(BMD 0 wt.%) it is about 20 days. It is attributedto the autocatalytic effect of carboxylic acid in thecopolymer on the degradation of nanoparticles That is, the rate of the degradation of core-formingblock was increased by the acidic catalyst in thehydrophobic block, which leads to a rapid collapseof nanoparticles. This result implies that the degra-dation rate of nanoparticles can be controlled by thecontent of carboxylic acid in the copolymer.
3.7. Drug release behavior The drug release behavior of nanoparticles was investigated using a dialysis membrane in phosphatebuffered (pH 7.4, 0.01 M, 37 jC). The concentration ofcopolymer containing the drug was fixed 0.5 wt.%. Asshown in nanoparticles without carboxylic acidexhibited a rapid release of 90% of drug within 10 h,whereas the nanoparticles containing carboxylic acidshowed controlled release of 50 – 90% for 7 days. In Fig. 6. DSC thermograms (a) and XPS spectra (b) of drug (PAP) addition, nanoparticles containing higher amounts of and PEO – PLA nanoparticles (BMD 19.5 wt.%) containing drug(14.9 wt.%).
carboxylic acid in the copolymer showed a much J. Lee et al. / Journal of Controlled Release 94 (2004) 323–335 was reported that the diffusion of methyl red wassubstantially more retarded in a toluene solution ofpoly(vinyl acetate) (PVAc) than in that of plystyrene(PS) The slower diffusion of MR in thepresence of a PVAc matrix was ascribed to thehydrogen bonding between the probe and polymer.
In our case, the release of drug from nanoparticleswas achieved by the diffusion of drug from the coreof nanoparticles to the aqueous medium because thenanoparticles are not degraded within the experimen-tal period. Thus, the hydrogen bonding between thecore-forming block and the drug retards the diffusionof drug, which plays an important role in the releaseof drug in nanoparticles.
Fig. 7. Release profiles of drug (PAP) from functionalized PEO – The PEO – PLA block copolymers containing a PLA nanoparticles in phosphate buffer solutions (pH 7.4, 0.01 M,37 jC). Solution concentration: 0.5 wt.%.
small quantity of carboxylic acid in the PLA blockwere successfully synthesized. The sizes of nano-particles prepared by a dialysis method are within slower release rate. This implied that the carboxylic the range of 30 – 40 nm, and they are suitable for an group of copolymer enhances the interaction between injectable drug carrier. The nanoparticles prepared nanoparticles and drug, leading to a decrease in the from the functionalized block copolymer have a very drug release rate and in the amount.
low CMC value, which suggests good stability of the Several mechanisms of drug release from biode- nanoparticles in spite of the presence of carboxylic gradable carriers have been proposed: Fickian acid. The drug loading efficiency of nanoparticles was diffusion through the polymer matrix, diffusion dramatically increased with the content of carboxylic through pores in the matrix, and drug liberation acid in the block copolymers. This result may be by polymer erosion. However, it is difficult to attributed to the hydrogen bonding between copoly- predict a drug release profile because this profile mer and drug. The release rate of drug was much is governed by various factors such as solubility of slower from nanoparticles containing higher amounts drug, degradation of polymer, and polymer – drug of carboxylic acid in the copolymer, which might be interaction. In this study, although the degradation associated with the enhanced interaction between the rate of nanoparticles is increased with the content carboxylic group of copolymers and the drug. These of carboxylic acid, the time required for degradation experimental results suggest that the nanoparticles is longer than 6 days even for nanoparticles con- prepared from functionalized PEO – PLA block taining BMD (19.5 wt.%). Therefore, it is specu- copolymers should be a good candidate for an inject- lated that the drug release from nanoparticles is able drug delivery carrier.
carried out mainly through diffusion within thisexperimental period.
Diffusion of a material in a polymer matrix is governed by the excluded volume, hydrodynamicinteraction, and specific interaction such as coulom- The authors thank the Ministry of Science and bic interaction and hydrogen bonding. When such a Technology of Korea (National Research Laboratory specific interaction takes place, the probe diffusion Project), POSTECH BSRI Research Fund, Pohang coefficient is affected significantly. For example, it Steel Company, and Advanced Environmental Bio- J. Lee et al. / Journal of Controlled Release 94 (2004) 323–335 technology Research Center for their financial lene oxide-co-h-benzyl L-aspartate) block copolymers: influ- ence of the poly(ethylene oxide) block on the conformation ofthe poly(h-benzyl L-aspartate) segment in organic solvents,Macromolecules 29 (1996) 3227 – 3231.
[14] C.S. Cho, J.W. Nah, Y.I. Jeong, J.B. Cheon, S. Asayama, H.
Akaike, T. Akaike, Conformational transition of nanoparticlescomposed of poly(g-benzyl L-glutamate) as the core and [1] C. Allen, D. Maysinger, A. Eisenberg, Nano-engineering poly(ethylene oxide) as the shell, Polymer 40 (1999) block copolymer aggregates for drug delivery, Colloids Surf., 6769 – 6775.
B Biointerfaces 16 (1999) 3 – 27.
[15] G.S. Kwon, T. Okano, Polymeric micelles as new drug car- [2] M.C. Jones, J.C. Leroux, Polymeric micelles—a new genera- riers, Adv. Drug Deliv. Rev. 21 (1996) 107 – 116.
tion of colloidal drug carriers, Eur. J. Pharm. Biopharm. 48 [16] M. Yokoyama, T. Okano, Y. Sakurai, K. Kataoka, Improved (1999) 101 – 111.
synthesis of adriamycin-conjugated poly(ethylene oxide) – [3] V.P. Torchilin, Structure and design of polymeric surfactant- poly(aspartic acid) block copolymer and formation of unimo- based drug delivery systems, J. Control. Release 73 (2001) dal micellar structure with controlled amount of physically 137 – 172.
entrapped adriamycin, J. Control. Release 32 (1994) 269 – 277.
[4] R. Nagarajan, K. Ganesh, Block copolymer self-assembly in [17] K. Taguchi, S. Yano, K. Hiratani, N. Minoura, Y. Okahata, selective solvents: theory of solubilization in spherical mi- Ring-opening polymerization of 3(s)-[(benzyloxycarbonyl) celles, Macromolecules 22 (1989) 4312 – 4325.
methyl]-1,4-dioxane-2,5-dione: a new route to a poly(alpha- [5] Z. Gao, A. Eisenberg, A model of micellization for block co- hydroxy acid) with pendant carboxyl groups, Macromolecules polymers in solutions, Macromolecules 26 (1993) 7353 – 7360.
21 (1988) 3338 – 3340.
[6] T. Nakanishi, S. Fukushima, K. Okamoto, M. Suzuki, Y. Mat- [18] G. Kwon, M. Naito, M. Yokoyama, T. Okano, Y. Sakurai, K.
sumura, M. Yokoyama, T. Okano, Y. Sakurai, K. Kataoka, Kataoka, Micelles based on AB block copolymers of poly Development of the polymer micelle carrier system for dox- (ethylene oxide) and poly(h-benzyl L-aspartate), Langmuir 9 orubicin, J. Control. Release 74 (2001) 295 – 302.
(1993) 945 – 949.
[7] M. Yokoyama, S. Fukushima, R. Uehara, K. Okamoto, K.
[19] Y. Kimura, K. Shirotani, H. Yamane, T. Kitao, Copolymeriza- Kataoka, Y. Sakurai, T. Okano, Characterization of physical tion of 3(s)-[(benzyloxycarbonyl) methyl]-1,4-dioxane-2,5-di- entrapment and chemical conjugation of adriamycin in poly- one and L-lactide: a facile synthetic method for functionalized meric micelles and their design for in vivo delivery to a solid bioabsorbable polymer, Polymer 34 (1993) 1741 – 1748.
tumor, J. Control. Release 50 (1998) 79 – 92.
[20] H.S. Yoo, T.G. Park, Biodegradable polymeric micelles com- [8] S.A. Hagan, A.G.A. Coombes, M.C. Garnett, S.E. Dunn, posed of doxoubicin conjugated PLGA – PEG block copoly- M.C. Davies, L. Illum, S.S. Davis, Polylactide – poly(ethylene mer, J. Control. Release 70 (2001) 63 – 70.
glycol) copolymers as drug delivery systems. 1. Characteriza- [21] A. Lavasanifar, J. Samuel, G.S. Kwon, The effect of fatty acid tion of water dispersible micelle-forming systems, Langmuir substitution on the in vitro release of amphotericin B from 12 (1996) 2153 – 2161.
micelles composed of poly(ethylene oxide)-block – poly(N- [9] P.L. Soo, L. Luo, D. Maysinger, A. Eisenberg, Incorporation hexyl sterarate-L-aspartamide), J. Control. Release 79 (2002) and release of hydrophobic probes in biocompatible polycap- 165 – 172.
rolactone-block – poly(ethylene oxide) micelles: implications [22] A. Lavasanifar, J. Samuel, G.S. Kwon, Poly(ethylene oxide)- for drug delivery, Langmuir 18 (2002) 9996 – 10004.
block – poly(L-amino acid) micelles for drug delivery, Adv.
[10] K. Cho, J. Lee, P. Xing, Enzymatic degradation of blends Drug Deliv. 54 (2002) 169 – 190.
of poly(q-caprolactone) and poly(styrene-co-acrylonitriles) [23] Z. Gan, T.F. Jim, M. Li, Z. Yuer, S. Wang, C. Wu, Enzymatic by Pseuomonas lipase, J. Appl. Polym. Sci. 83 (2002) biodegradation of poly(ethylene oxide-b – q-caprolactone) di- 868 – 879.
block copolymer and its potential biomedical applications, [11] C. Allen, Y. Yu, D. Maysinger, A. Eisenberg, Polycaarpolac- Macromolecules 32 (1999) 590 – 594.
tone-b – poly(ethylene oxide)block copolymer micelles as a [24] R. Jalil, J.R. Nixon, Biodegradable poly(lactic acid) and po- novel drug delivery vehicle for neurotrophic agents FK506 ly(lactide-co-glycolide) microcapsules: problems associated and L-685, 813, Bioconjug. Chem. 9 (1998) 564 – 572.
with preparative techniques and release properties, J. Micro- [12] S.B. La, T. Okano, K. Kataoka, Preparation and characteriza- encapsul. 7 (1990) 297 – 325.
tion of micelle-forming polymeric drug indomethacin-incor- [25] J. Lee, K. Park, T. Chang, J.C. Jung, Polymer/probe interac- porated poly(ethylene oxide) – poly(h-benzyl L-aspartate) tion in probe diffusion through a polymer matrix: methyl red block copolymer micelles, J. Pharm. Sci. 85 (1996) 85 – 90.
diffusion in poly(vinyl acetate)/toluene solutions, Macromole- [13] S. Cammas, A. Harada, Y. Nagasaki, K. Kataoka, Poly(ethy- cules 25 (1992) 6977 – 6979.

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