Poly( n -butylcyanoacrylate) Submicron Particles Loaded with Ciprofloxacin for Potential Treatment of Bacterial Infections

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Poly(n-butylcyanoacrylate) submicron colloidal particles loaded with the antibiotic ciprofloxacin are prepared by emulsion polymerization and characterized by scanning electron microscopy, dynamic light scattering, nuclear magnetic resonance and
      U   n  c  o   r   r  e  c   t  e   d P   r  o  o   f    U   n  c  o   r   r  e  c   t  e   d P   r  o  o   f 1  Progr Colloid Polym Sci (2010) 137:53–59 2  DOI: 10.1007/2882_2010_11 3  Poly( n -butylcyanoacrylate) submicron particles loaded 4  with ciprofloxacin for potential treatment of bacterial infections 5  Georgi Yordanov 1 , Nikola Abrashev 2 , and Ceco Dushkin 1 6  Abstract  Poly( n -butylcyanoacrylate) submicron colloidal 7  particles loaded with the antibiotic ciprofloxacin are 8  prepared by emulsion polymerization and characterized by 9  scanning electron microscopy, dynamic light scattering, nu- 10  clear magnetic resonance and gel-permeation chromatogra- 11  phy. The entrapment efficiency for ciprofloxacin in the 12  polymer particles is investigated as a function of the mono- 13  mer and drug concentrations in the polymerization medium. 14  The kinetics of ciprofloxacin release from the polymer par- 15  ticles is found to depend on the pH of the release medium. 16  Studies by gel-permeation chromatography indicate for a 17  possible drug-polymer association. The antibacterial activity 18  of the obtained formulation is tested on a clinical isolate of  19  Escherichia coli  bacterium and is found to be similar to that 20  of the free drug. 21  Keywords  poly( n -butylcyanoacrylate) • colloids • submi- 22  cron particles • ciprofloxacin • drug release •  Escherichia 23  coli 24  Introduction 25  The development of various methods for targeted delivery of  26  antibiotics to infected cells and foci of bacterial infection 27  represents a hot topic in drug delivery research. The use of  28  colloidal submicron particles as drug carriers [1-4] is an 29  alternative approach to the classical antibacterial therapy. 30  It consists of association of the drug to a submicron particle 31 carrier, thereby hiding and protecting the antibiotic molecule 32 from degradation, and its delivery to inaccessible target cells 33 in a controlledmanner, which may be beneficial for the treat- 34 ment of intracellular infections [5, 6]. In this regard, poly 35 (alkylcyanoacrylate) (PACA) submicron particles meet ide- 36 ally the requirements for antibiotic-carrier systems due to a 37 number of advantageous properties such as biocompatibility, 38 biodegradability, and low toxicity [7-15]. The entrapment of  39 antibiotics in PACA submicron particles has been found to 40 increase significantly the drug therapeutic efficiency against 41 intracellular pathogens [8-10]. 42 On the other hand, ciprofloxacin (CIP) is a fluoroquino- 43 lone antibiotic with a broad activity against a variety of  44 clinically relevant microorganisms. It is used for the treat- 45 ment of urinary tract infections, gastrointestinal infections, 46 sexually transmitted infections, as well as infections of the 47 skin and bones [16]. Promising formulations have been 48 previously prepared by the association of CIP with human- 49 serum-albumin colloidal particles [17], poly(D,L-lactide- co - 50 glycolide) submicron particles [18-20], as well as submicron 51 particles of poly(isobutylcyanoacrylate) (PIBCA) [21-23] 52 and poly(ethylbutylcyanoacrylate) (PEBCA) [24]. 53 Here we report on the preparation, physicochemical 54 characterization and the antibacterial activity of ciprofloxa- 55 cin-loaded poly( n -butylcyanoacrylate) (PBCA) submicron 56 particles intended for the treatment of bacterial infections. 57 We demonstrate the successful entrapment of CIP in PBCA 58 submicron particles by emulsion polymerization using the 59 surfactant PEG-PEO-PEG amphiphilic triblock copolymer  60 (Pluronic F68). Previous studies on the entrapment of CIP in 61 PIBCAparticlesarebasedondispersionpolymerizationusing 62 the colloidal stabilizer dextran 70 [21,22]. The dispersion 63 polymerization of alkylcyanoacrylates proceeds in a different 64 way than the emulsion polymerization (see the discussion 65 section). On the other hand, the previously reported studies 66 on the entrapment of CIP in PEBCA submicron particles 67 have found it being possible only in the presence of acetone 68 (30 %, v/v) in the polymerization medium [24]. These results 69 indicate that small changes in the formulation may lead to a G. Yordanov ( * ) 1 Laboratory of Nanoparticle Science and Technology, Departmentof General and Inorganic Chemistry, Faculty of Chemistry, SofiaUniversity ‘‘St. Kliment Ohridski’’, 1 “James Bourchier” Blvd., 1164,Sofia, Bulgariae-mail: chem_gbg@yahoo.com 2 Microbiological Laboratory, Multiprofile Hospital for Active Treat-ment ‘‘Dr. Nikola Vasiliev’’, 1 “17-th January” Sqr., 2500 Kyustendil,Bulgaria # Springer-Verlag Berlin Heidelberg 2010  53      U   n  c  o   r   r  e  c   t  e   d P   r  o  o   f 70  different behavior, depending on the type of monomer used. 71  The synthesis of CIP-loaded PBCA submicron particles 72  reported here is acetone-free, which is more advantageous 73  for potential pharmaceutical applications; nevertheless the 74  n -butylcyanoacrylate monomer is approved for human use 75  as surgical glue [25]. We characterize the obtained formula- 76  tion by scanning electron microscopy, dynamic light scat- 77  tering, nuclear magnetic resonance, and gel-permeation 78  chromatography. The effects of monomer and drug concen- 79  trations in the polymerization medium on the entrapment 80  efficiency for CIP are investigated. The effect of pH on the 81  drug release kinetics and particle erosion is studied. Antibac- 82  terial susceptibility tests of the obtained formulation against a 83  clinicalisolate of   Escherichiacoli bacteriumare performed.It 84  is found that the entrapped drug is of nearly the same efficien- 85  cy as the free one, which opens the way of its application 86  bearing in mind the other advantages of polymer submicron 87  particles. 88  Experimental procedures 89  Materials and reagents.  The  n -butylcyanoacrylate (BCA) 90  monomer was from Special Polymers Ltd (Bulgaria). Cipro- 91  floxacin (CIP) lactate was from KRKA (Slovenia). Phos- 92  phate-buffered saline (PBS; various pH values were 93  adjusted by 1 M HCl), citric acid (anhydrous), sodium 94  hydroxide (puriss. p.a.,  > 99 %), hydrochloric acid (37 %), 95  and Pluronic F68 were from Sigma. Glucose (10 %, w/w) 96  was from Actavis (Bulgaria). 97  Preparation of ciprofloxacin-loaded submicron particles. 98  The polymerization medium was prepared by dissolving 99  Pluronic F68 (20 mg) and citric acid (20 mg) in glucose 100  solution (5 ml 10 %, w/w); then ciprofloxacin (1-3 mg/ml) 101  was added and diluted with distilled water to 10 ml. The 102  n -butylcyanoacrylate monomer (50-200  m l) was added drop- 103  wise to the polymerization medium upon vigorous stirring 104  (  600 rpm rotation speed). After polymerization (for 3 105  hours), the pH of the obtained colloidal dispersion was 106  adjusted at 5.6 by the addition of 1 M NaOH (0.2 ml). Pure 107  PBCA particles were prepared as a reference at similar  108  conditions but without addition of CIP. The amount of  109  particles in the as-obtained dispersion was determined by 110  centrifugation (14500 rpm, 30 min) of an aliquot (1 ml) from 111  the dispersion in a pre-weighted Eppendorf tube, followed 112  by washing with distilled water and vacuum drying. The as- 113  obtained drug-loaded particles can be stored at 4  C for at 114  least one month without any observable changes. 115  Characterization of the submicron particles.  The 116  as-obtained submicron particles were imaged by a scanning 117  electron microscope (SEM) JSM-5510 (JEOL). Dynamic 118  light scattering (DLS) system Malvern 4700C (Malvern 119 Instruments, UK) was used to measure the particle size and 120 size distribution (each value was obtained as average of five 121 measurements). Nuclear magnetic resonance ( 1 H-NMR) 122 spectra were taken in acetone-d6 with Bruker Avance II þ 123 600 spectrometer (spectrometer frequency 600.13 MHz). 124 The molecular weight of polymer was determined by gel- 125 permeation chromatography (GPC) using a GPC system 126 with refractive index (RI Waters M410) and ultra-violet 127 (UV Waters M484) detectors, operating at a wavelength of  128 254 nm. Styragel columns with nominal pore sizes of 100 129 and 500 A˚were utilized. Tetrahydrofuran (THF) was used as 130 the eluent, with a flow rate of 1 ml/min, at 45  C. The 131 samples were prepared as solutions in THF (3.5 mg/ml). 132 The calibration was carried out with poly(ethylene glycol) 133 (PEG) standards. The CIP-PBCA particles used for the 134 NMR and GPC analyses were purified from the unloaded 135 drug by centrifugation, washed twice with distilled water, 136 and dried under vacuum. 137  Drug entrapment efficiency.  The amount of CIP 138 entrapped in PBCA particles was determined by centrifuga- 139 tion of the as-prepared colloid dispersions (14500 rpm, 140 20 min) and spectrophotometrical determination of CIP in 141 the supernatant. The measurements are made in 0.01 M PBS 142 (pH 7.4) by a double-beam UV-vis spectrophotometer  143 Evolution 300 (Thermo Scientific) at 334 nm. The entrap- 144 ment efficiency is defined as the fraction of drug entrapped 145 in the polymer particles with respect to its total amount. 146  Drug release kinetics and particle erosion.  An aliquot 147 (1 ml) of the as-prepared particle dispersions was centri- 148 fuged (14500 rpm, 10 min) to remove the non-entrapped 149 drug; the particles were washed with 0.2 % (w/v) aqueous 150 solution of Pluronic F68 (1 ml) and re-dispersed in PBS 151 (1 ml) by ultrasound sonication for 3 min. After that the 152 particles were transferred into 49 ml of the release medium 153 (0.01 M PBS, pH 4.6, 5.6, 6.5 or 7.4). The release buffer  154 contained also 0.2 % (w/v) Pluronic F68 in order to avoid 155 any particle aggregation during the release experiments. The 156 experiments were carried out at 37  C in a closed vessel (to 157 prevent evaporation of the release medium) upon magnetic 158 stirring (300 rpm). The amount of released drug was 159 determined by taking aliquots from the release medium, 160 removing the dispersed particles by centrifugation, and spec- 161 trophotometrical measurement of the drug concentration in 162 the obtained supernatant. The particle erosion was evaluated 163 by spectrophotometric measurements of the optical density 164 at 400 nm as previously described [24] (actually, the light 165 scattering by the particles in the dispersion causes a reduc- 166 tion in the light transmission). 167  Antibacterial susceptibility tests.  The antibacterial activ- 168 ities of free ciprofloxacin, CIP-PBCA particles and pure 169 PBCA particles were compared by a dilution susceptibility 170 test in Mueller-Hinton broth (Merck, Germany) on a cipro- 171 floxacin-susceptible strain of   Escherichia coli . The strain was 54 G. Yordanov et al.      U   n  c  o   r   r  e  c   t  e   d P   r  o  o   f 172  a clinical isolate obtained from infected wound, identified and 173  biochemically characterized as indole ( þ ), citrate (  ), urease 174  (  ), lysine decarboxylase ( þ ) and ornithine decarboxylase 175  ( þ ). The activity of the CIP-PBCA formulation was com- 176  pared with that of a CIP solution with the same drug concen- 177  tration. The minimal inhibitory concentration (MIC) was 178  defined as the lowest antibiotic concentration inhibiting bac- 179  terial growth (determined by measuring the optical density) 180  after incubation for 18 h at 37  C. 181  Results and discussion 182  The PBCA colloidal particles can be prepared by two differ- 183  ent procedures: (i) emulsion polymerization, and (ii) disper- 184  sion polymerization. The emulsion polymerization of  185  alkylcyanoacrylates proceeds in a different way in compari- 186  son with the classical emulsion polymerization [26-28]. It 187  consists of the dropwise addition of monomer to acidic 188  aqueous solution of a suitable surfactant (such as Pluronic 189  F68). At these conditions the micelles take up monomers 190  resulting in swollen micelles. The hydroxide anions initiate 191  polymerization into the swollen micelles via anionic mecha- 192  nism. The further growth of particles is a result of monomer  193  transfer from the larger droplets to the initially formed 194  primary particles. During this process, drug molecules can 195  be entrapped between the polymer chains. Furthermore, 196 various non-covalent interactions (such as hydrogen bonds, 197 electrostatic attraction, etc.) may increase the drug associa- 198 tion with the polymer. For example, it is well known that 199 PACA colloidal particles adsorb on their surface various 200 cationic drugs [21,23,29]. 201 The difference in the case of dispersion polymerization is 202 thatcolloidalstabilizers (suchasdextrans)are usedinsteadof  203 surfactants. At these conditions, the hydroxide anions initiate 204 the reaction to form PACA oligomers resulting in the forma- 205 tion of the primary polymer particles by precipitation. These 206 initial particles add further monomers and oligomers thus 207 growing in size. The colloidal stabilizer prevents the particle 208 aggregation. Dextran 70 has been previously used as a stabi- 209 lizer for the preparation of CIP-loaded PEBCA colloids, but 210 the drug entrapment is possible only in the presence of ace- 211 toneinthiscase[24].Ourpreliminarytestsonthepreparation 212 of CIP-PBCA particles by dispersion polymerization showed 213 that the utilization of dextran results in relatively unstable 214 colloids, which coagulate soon after their preparation. In 215 contrast, the emulsion polymerization in the presence of  216 Pluronic F68 results in the formation of stable CIP-PBCA 217 colloids, whose characteristics are described below. 218 The SEM shows that the as-obtained CIP-PBCA particles 219 are spherical in shape of an average size of about 240 nm 220 (Fig. 1a). It is interesting that the pure PBCA particles 221 prepared at similar conditions are much smaller – about 222 170 nm in diameter (Fig. 1b). The procedure for sample 100 200 300 400 500 60001020304050 a    %    i  n  c   l  a  s  s size, nm10020030040050060001020304050    %    i  n  c   l  a  s  s size, nm   b Fig. 1  Representative SEMimages ( left  ) and the respectivesize-distributions obtained byDLS ( right  ) of CIP-PBCAparticles (a) and pure PBCAparticles (b). The particlesare prepared with n -butylcyanoacrylateconcentration 10 mg/ml. The CIPconcentration is 3 mg/ml in thecase of CIP-PBCA particles. Poly( n- butylcyanoacrylate) submicron particles loaded with ciprofloxacin for potential treatment of bacterial infections 55      U   n  c  o   r   r  e  c   t  e   d P   r  o  o   f 223  preparation (centrifugation, washing twice with distilled 224  water and drying on a glass substrate under vacuum) results 225  in a partial aggregation of the particles on the glass substrate. 226  However, the DLS analyses demonstrate that there are no 227  particle aggregates in the as-obtained colloidal dispersions. 228  The particle size distribution is monomodal as confirmed by 229  both SEM and DLS. 230  The reason for different sizes of CIP-PBCA and pure 231  PBCA particles is unclear. May be it is a result of the 232  different molecular weight of polymer (see below), and/or  233  more complex effect of CIP on the emulsion polymerization 234  process. It is important to note that not the entire amount of  235  initial monomer is transformed into PBCA particles. For  236  example, the particle content in the dispersion of pure 237  PBCA (prepared with initial monomer concentration 10 238  mg/ml) is only 6.9 mg/ml, which means that about 30 % of  239  the initial monomer is not included in the obtained particles. 240  Similarly, the particle content in the respective dispersion of  241  CIP-PBCA is 6.3 mg/ml (the values of particle content are 242  usually scattered with   10 %). The monomer that is not 243  included into polymer particles may form oligomers with 244  lower molecular weight, which are probably solubilized in 245  the polymerization medium containing the surfactant. Also, 246  a small part of polymer (usually below 5-10 % of the initial 247  monomer) is found deposited on the Teflon-coated stirring 248  bar after the particle preparation. 249  The successful entrapment of CIP in PBCA particles is 250  confirmed by  1 H-NMR spectroscopy. The signals for methy- 251  lene protons appear in the spectrum of pure PBCA at 1.5 ppm 252  (2H), 1.7 ppm (2H), 2.9 ppm (2H) and 4.3 ppm (2H). The 253  signal at 1.0 ppm (3H) is from methyl protons. Signals from 254  the entrappedCIP molecules appear at8.74ppm(1H,s), 7.94 255  ppm(1H,d,  J  ¼ 13.27Hz)and7.72ppm(1H,d,  J  ¼ 7.30Hz). 256  Since the drug is water-soluble and is used below its 257  solubility, a question arises about the mechanism of its 258  loading in the relatively hydrophobic PBCA particles. 259  There are few possible forms of drug association with the 260  polymer. First, the drug may form electrostatic interactions 261  with the surface of the polymer particles. Previous studies 262  demonstrated that CIP could be adsorbed on PEBCA col- 263  loids resulting in remarkable reduction of the zeta potential 264  [24]. Similarly, other water-soluble drugs (such as doxoru- 265  bicin, methotrexate, dactinomycin, etc.) could be adsorbed 266  on the surface of PACA particles [23,29]. Second, CIP could 267  be entrapped in the polymer matrix of the particles in its free 268  base form (neutral molecule), which is less polar than the 269  ionic forms. Indeed, on the basis of   19 F-NMR analysis, the 270  CIP loaded in PEBCA has been previously found only in its 271  free base form [24]. Finally, some CIP molecules could be 272  modified by the addition of alkylcyanoacrylate monomers 273  (see below), which may result in increased affinity toward 274  the polymer material. 275 The polymerization of alkylcyanoacrylates in aqueous 276 media usually proceeds via anionic mechanism (the reaction 277 is initiated by OH - ) [26-28]. Previous investigations 278 indicated that the CIP molecules could initiate zwitterionic 279 polymerization of alkylcyanoacrylates via the amino-group, 280 thus becoming covalently bonded to the polymer backbone 281 [24]. Since the nitrogen from the piperazine amino-group 282 can provide an electron pair, a nucleophilic attack occurs 283 leading to the formation of a covalent bond between the CIP 284 molecule and the  n -BCA monomer, followed then by the 285 polymerization. We assume that a similar process may take 286 place during the CIP entrapment in PBCA particles, reported 287 here. It is difficult to use the data from  1 H-NMR in order to 288 confirm a possible association/interaction between CIP 289 molecules and the polymer. Therefore, we use gel perme- 290 ation chromatography (GPC) with a dual simultaneous 291 detection (UV and RI) to obtain information about 292 two important characteristics of the CIP-PBCA colloids: 293 the molecular weight of PBCA polymer; and indication of  294 association/interaction between the polymer and the CIP 295 molecules. 296 The molecular weights obtained in our experiments are typi- 297 cal for PBCA colloids prepared by emulsion polymerization 02040608010012022 23 24 25Time, min    A   b  s  o  r   b  a  n  c  e  a   t   2   5   4  n  m ,  m   A   U 2530354045    R  e   f  r  a  c   t   i  v  e   I  n   d  e  x ,      µ    R   I Fig. 2  GPC chromatograms of CIP-loaded PBCA particles dissolvedin THF. The chromatograms are recorded with double (UV and RI)simultaneous detection. t1 : 1 Table 1  Results from the GPC analysis of polymer from CIP-PBCAand pure PBCA particles. The particles are prepared using c(BCA) ¼ 10mg/ml and c(CIP) ¼ 2 mg/mlParticles DetectionMax RT,min Mp Mw Mz Mn PD  t1 : 2 CIP-PBCA UV-254 22.94 1521 1502 1519 1485 1.01  t1 : 3 RI 23.72 838 862 903 818 1.05  t1 : 4 PBCA UV-254 22.95 1514 1688 2431 1436 1.31  t1 : 5 RI 22.95 1272 2205 2890 1667 1.32  t1 : 6t1 : 7 RT – retention time; Mw = weight-average molecular weight; Mn =number-average molecular weight; Mz ¼ Z-average molecular weight;Mp ¼ peak molecular weight; PD ¼ Mw/Mn 56 G. Yordanov et al.      U   n  c  o   r   r  e  c   t  e   d P   r  o  o   f 298  [30-34] (Table 1). The GPC chromatograms of CIP-PBCA 299  particles dissolved in THF are shown in Fig. 2. As seen, 300  there are two fractions of oligomeric molecules in the 301  case of CIP-PBCA: (i) a ‘‘heavier’’ fraction, with Mw 302   1500, which shows intensive UV-absorption at 254 nm; 303  and (ii) a ‘‘lighter’’ fraction, with Mw   860, which shows 304  very low UV-absorption. The refractive index (RI) detection 305  indicates that fraction (ii) is the predominant one. The PBCA 306  polymer is not expected to show a significant light absor- 307  bance at 254 nm due to the lack of a chromophore group. 308  However, the CIP molecule contains an aromatic ring, 309  which is a UV-chromophore. We suppose that the stronger  310  UV-absorption of fraction (i) corresponds to PBCA asso- 311  ciated with CIP molecules, while fraction (ii) contains pure 312  PBCA oligomers. The GPC analysis of pure PBCA, carried 313  out at similar conditions, indicates the formation of longer  314  oligomeric chains (14-mers) with Mw   2200 (measured 315  by using RI-detector). The higher molecular weight in the 316  case of pure PBCA could be a result of the slightly lower  317  pH (2.52) of the polymerization medium (the polymerization 318  medium, which contains CIP has a pH  ¼  2.86). This is 319  expected to result in the initiation of smaller number of  320  growing polymeric chains (the initiator is OH - , whose 321  concentration is lower in more acidic pH). Interestingly, 322  the polydispersity index (PD) of the obtained polymer in 323  the case of CIP-PBCA is smaller (1.05) than in the case of  324  pure polymer (1.32). It means that the molecular weight 325  distribution of the polymer in the case of CIP-PBCA 326  is appreciably narrower than that of pure PBCA. Similar  327  difference in the PD-values has been previously observed 328  in the case of CIP-loaded poly(ethylbutylcyanoacrylate) 329  colloids [24]. 330  The effect of initial concentration of CIP in the polymer- 331  ization medium on the drug entrapment efficiency is shown 332  in Fig. 3a. In these experiments, the concentration of  333  n -BCA is kept constant at 10 mg/ml. As seen, higher entrap- 334  ment efficiency is achieved at lower CIP concentrations 335  (  1 mg/ml). At higher CIP concentrations the entrapment 336  efficiency decreases. The initial  n -BCA concentration in the 337 polymerization medium (at a constant CIP concentration 338 2 mg/ml) has a different effect on the drug entrapment in 339 particles, as seen from Fig 3b. Increasing the monomer  340 concentration increases the drug entrapment efficiency. 341 Interestingly, we observed variations in the entrapment 342 efficiency (about   10 %) depending on the batch of  343 n -BCA used. In order to avoid confusion from such batch- 344 to-batch variations,  n -BCA from the same batch is used for  345 evaluation of the effects shown in Fig. 3. As an optimal 346 formulation we find the CIP-PBCA colloidal particles, 347 prepared at CIP and  n -BCA concentrations 3 mg/ml and 10 348 mg/ml, respectively. These particles are used for further  349 studies of the drug release kinetics and antimicrobial activity. 350 Since submicron particles could target intracellular lyso- 351 somal compartments (following phagocytosis by macro- 352 phages) [5,6,8-10], it is important to investigate the drug 353 release kinetics at various pH (the pH of blood is about 354 7.4, but inside lysosomes it is about 4.6). Since, the CIP 355 molecule contains both amino (NH) and carboxylic (COOH) 356 groups, it is a typical zwitterionic drug, which exists in 357 aqueous solutions of CIP in different forms depending on 358 the pH. The pKa values for the COOH and NH 2+ groups are 359 5.86 (pKa 1 ) and 8.24 (pKa 2 ), respectively [35,36]. By using 360 these values one can calculate the distribution of the differ- 361 ent CIP forms (cationic, neutral and anionic) at given pH. 362 At pH  <  5 more than 90 % of CIP exists as a cation. At 363 physiological pH (7.4) CIP is in its neutral form (zwitterion 364 and molecule). Calibration curves for spectrophotometric 365 determination of CIP concentration at different pH are 366 built (the UV-vis absorbance spectrum of CIP depends on 367 the pH of solution). Parallel to the drug release experiments, 368 controls of pure PBCA particles dispersed in the release 369 medium for a different time are performed for a baseline 370 reference in the measurements of CIP concentration. The 371 drug release profiles from PBCA nanoparticles at various pH 372 of the release medium are shown in Fig. 4a. The highest 373 release rate is observed at pH 7.4. The release rate is lowest 374 at pH  ¼  5.6. At the same time, the particle erosion is 375 followed indirectly by measurements of the optical density 253035404550 a b 0 1 2 3 4CIP concentration, mg/ml    E  n   t  r  a  p  m  e  n   t  e   f   f   i  c   i  e  n  c  y ,   % 2025303540450 10 20 30BCA concentration, mg/ml    E  n   t  r  a  p  m  e  n   t  e   f   f   i  c   i  e  n  c  y ,   % Fig. 3  Drug entrapmentefficiency as a functions of (a) CIP concentration (at constant n -BCA concentration 10 mg/ml),and (b)  n -BCA concentration(at constant CIP concentration2 mg/ml). All experiments aremade using  n -BCA from thesame batch. Poly( n- butylcyanoacrylate) submicron particles loaded with ciprofloxacin for potential treatment of bacterial infections 57
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