Assessment of the Integrity of Poly(caprolactone)-b-poly(ethylene

Here we evaluate the integrity of poly(caprolactone)-b-poly(ethylene oxide) micelles in media of varying chemical complexity and in cells by using flu...
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Langmuir 2006, 22, 3570-3578

Assessment of the Integrity of Poly(caprolactone)-b-poly(ethylene oxide) Micelles under Biological Conditions: A Fluorogenic-Based Approach Radoslav Savic´, Tony Azzam, Adi Eisenberg, and Dusica Maysinger* Department of Pharmacology and Therapeutics, McGill UniVersity, Montreal, Quebec, Canada H3G 1Y6, and Chemistry Department, McGill UniVersity, Montreal, Quebec, Canada H3A 2K6 ReceiVed NoVember 26, 2005. In Final Form: January 24, 2006 The integrity of block copolymer micelles is important for their effectiveness and successful delivery of the incorporated drugs. Here we evaluate the integrity of poly(caprolactone)-b-poly(ethylene oxide) micelles in media of varying chemical complexity and in cells by using fluorogenic micelles. Fluorogenic dye fluorescein-5-carbonyl azide diacetate was covalently attached to the micelle-core-forming part of the block copolymer, poly(caprolactone). The fluorescence was not detectable unless the poly(caprolactone)21-b-poly(ethylene oxide)45 micelles were destroyed and the fluorogenic dye was activated by deesterification. The fluorescence of the activated dye from destroyed micelles was easily detectable in various media and in cells. Micelles were stable in simple media such as phosphate-buffered saline but disassembled to varying extents with increasing chemical complexity of the media and addition of serum. The integrity of the internalized micelles within the cells showed a time-dependent decrease but remained largely preserved (80%) after 20 h of incubation with cells. A proof of principle was also demonstrated in vivo in mice. The fluorogenic approach to micelle integrity assessment presented herein should lend itself to other block copolymer micelles and assessments of their integrity in complex biological systems in vitro and in vivo.

Introduction Block copolymer micelles are nanosized particles with a typical core-shell structure. The core solubilizes the hydrophobic drugs, and the corona allows the suspension of micelles in an aqueous medium.1-3 The use of block copolymer micelles as drug-carrying vehicles was proposed by Ringsdorf’s group in the 1980s.4-6 The rationale for incorporating small-molecular-weight drugs in micelles includes overcoming common drug problems such as poor solubility in water, toxic side effects, and poor pharmacokinetics.1,2,7 Micelles enable up to several thousand times greater amounts of micelle-incorporated drugs to be administered in aqueous media, and micelles also change the pharmacokinetics of micelleincorporated drugs. Whereas free drugs are usually cleared from circulation within minutes of intravenous administration, micelles can circulate for hours and accummulate in sites of leaky vasculature, carrying along the drug that is solubilized within the micelles. However, if micelles were to disassemble prematurely, then the incorporated hydrophobic drug would be immediately released and precipitated. Hence, increased drug accumulation of micelle-incorporated drugs in tumors, their most investigated application, would be adversly affected by micelle disassembly and premature drug loss from micelles. Therefore, a critical issue for the effectiveness * Corresponding author. E-mail: [email protected]. (1) Adams, M. L.; Lavasanifar, A.; Kwon, G. S. J. Pharm. Sci. 2003, 92, 1343-1355. (2) Kabanov, A. V.; Alakhov, V. Y. Crit. ReV. Ther. Drug Carrier Syst. 2002, 19, 1-72. (3) Riess, G. Prog. Polym. Sci. 2003, 28, 1107-1170. (4) Hirano, T.; Klesse, W.; Ringsdorf, H. Macromol. Chem. Phys. 1979, 180, 1125-1131. (5) Ringsdorf, H. Abstracts of Papers of the American Chemical Society; 1980; Vol. 180, 46-ORPL. (6) Gros, L.; Ringsdorf, H.; Schupp, H. Angew. Chem., Int. Ed. Engl. 1981, 20, 305-325. (7) Yokoyama, M. In Biorelated Polymers and Gels: Controlled Release and Applications in Biomedical Engineering; Okano, T., Ed.; Academic Press: San Diego, CA, 1998; Chapter 6, pp 193-229.

of micelle-incorporated drugs is the micelle integrity, and approaches to enhance the stability of micelles include cross linking of the micelle core/corona 8,9 and the preparation of stereocomplex micelles.10 However, information on the micelle integrity in the presence of biological media, and most notably in cells, is scarce to nonexistent. This is at least in part due to limitations of commonly used microscopic, spectroscopic, and chromatographic approaches3,11,12 of micelle characterization. These methods are less adequate in studying the integrity of block copolymer micelles in biological media, which may contain aggregates of, for example, proteins, lipids, or phospholipids.2 Understanding the fate of micelles and micelle-incorporated drugs under biologically relevant conditions is necessary to facilitate the development of advanced, controlled micellar drug delivery vehicles and realize the full potential of micelles in drug delivery. In the present study, we investigate the integrity of PCL21-b-PEO45 micelles in various media, cells, and in vivo by using a fluorogenic-based approach to micelle integrity assessment. Materials and Methods Monomethoxy-poly(ethylene glycol) with an average molecular weight of 2000 (Mw/Mn ) 1.07) was from Fluka, stannous(II) octoate was from Sigma, and -caprolactone was from Aldrich. F-5-CADA was from Molecular Probes. The poly(ethylene glycol) macroinitiator was dried by azeotropic distillation in toluene followed by vacuum at 50 °C for 24 h. -Caprolactone was dried twice over CaH2 and distilled under reduced pressure just before use. Toluene was refluxed over sodium/benzophenone-ketyl and distilled under a nitrogen (8) Joralemon, M. J.; O’Reilly, R. K.; Hawker, C. J.; Wooley, K. L. J. Am. Chem. Soc. 2005, 127, 16892-16899. (9) Miyata, K.; Kakizawa, Y.; Nishiyama, N.; Harada, A.; Yamasaki, Y.; Koyama, H.; Kataoka, K. J. Am. Chem. Soc. 2004, 126, 2355-2361. (10) Kang, N.; Perron, M. E.; Prud’homme, R. E.; Zhang, Y. B.; Gaucher, G.; Leroux, J. C. Nano Lett. 2005, 5, 315-319. (11) Magenheim, B.; Benita, S. STP Pharma Sci. 1991, 1, 221-241. (12) Sastry, N. V.; Hoffmann, H. Colloids Surf., A 2004, 250, 247-261.

10.1021/la0531998 CCC: $33.50 © 2006 American Chemical Society Published on Web 03/09/2006

Poly(caprolactone)-b-poly(ethylene oxide) Micelles atmosphere. All other chemicals and solvents were of analytical grade and were used as received. All tissue culture plates were from Sarstedt, and tissue culture media were from GIBCO. Synthesis of PCL-b-PEO Block Copolymer. PCL-b-PEO block copolymer was synthesized according to Bogdanov.13 Briefly, 6.0 mL of freshly distilled -caprolactone (54 mmol) was introduced (under nitrogen) into a flask containing 5 g (2.5 mmol) of dried monomethoxy-poly(ethylene glycol). The mixture was freezethawed three times to remove dissolved oxygen, followed by the addition of stannous(II) octoate (0.01 wt %). Next, the flask was placed in liquid nitrogen, evacuated for 1 h, sealed off, and vigorously stirred at 135 °C for 24 h. The resulting viscous crude (cooled to room temperature) was dissolved in tetrahydrofuran (50 mL) and precipitated by dropwise addition into cold hexanes. The white precipitate was collected by filtration and dried at 40 °C for 2 days under vacuum (yield: 10.6 g). The degrees of polymerization and polydispersity were measured by size-exclusion chromatography in tetrahydrofuran. A Waters 510 liquid chromatography pump equipped with two (HR1 and HR4) serially connected Styragel columns and a Varian RI-4 refractive index detector were used at room temperature. Poly(styrene) standards were used for calibration. NMR spectra were recorded on a Varian XL-300 spectrometer. CDCl3 and d6-DMSO were used as solvents, and TMS was used as an internal reference. Using poly(styrene) standards and size-exclusion chromatography, we determined the number-average molecular weight of starting PEO (before reacting with caprolactone monomer) to be Mn ) 2960 (polydispersity 1.05), and after the reaction, we determined the value for PCL-b-PEO to be Mn ) 6960 (polydispersity 1.40). 1H NMR (CDCl3): 1.3-1.75 (m, CH3O (CH2CH2O)45-(COCH2(CH2)3CH2O)21H); 2.25-2.4 (t, CH3O(CH2CH2O)45-(COCH2(CH2)3CH2O)21H); 3.38 (s, CH3O(CH2CH2O)45-(COCH2(CH2)3CH2O)21H); 3.6-3.8 (m, CH3O (CH2CH2O)45-(COCH2(CH2)3CH2O)21H); and 4.0-4.1 (t, CH3O(CH2CH2O)45-(COCH2 (CH2)3CH2O)21 H) ppm. On the basis of the integration values from NMR and the degree of polymerization of the narrow polydisperse PEO block (45), the degree of polymerization of the PCL block was estimated to be 21. This is in agreement with the starting feed (22), which suggests that over 95% of the -caprolactone monomer was reacted. Synthesis of F-5-CADA-Labeled PCL-b-PEO. Twenty milligrams of PCL21-b-PEO45 (4.6 µmol) were dissolved in a small amount of freshly distilled benzene and freeze dried for 6 h. Freeze drying was repeated twice to remove traces of water. The polymer and F-5-CADA (10 mg, 20 µmol) were dissolved in 5 mL of freshly distilled toluene. The mixture was protected from light, heated (80 °C) for 6 h, and left overnight at ambient temperature. Next, the solvent was removed under reduced pressure, and the resulting crude was dissolved in tetrahydrofurane (0.5 mL) and applied to a silica gel column (10 g) preswollen with hexanes. The column was eluted with ethyl acetate/hexanes (80/20) until unreacted dye was no longer detectable in the eluate by thin-layer chromatography. The column was next eluted using methanol/dichloromethane with a gradual increase in methanol from (1/99) to (5/95). The fractions containing F-5-CADA-PCL21-b-PEO45 were collected, and the solvent was removed under reduced pressure. The polymer was dissolved in dioxane, and a colorless powder of labeled PCL-b-PEO was recovered by freeze drying. The overall yield, relative to the PCL-b-PEO polymer, was 50%. Purification of Labeled PCL-b-PEO. The crude F-5-CADAPCL-b-PEO was eluted with a mixture of ethyl acetate/hexanes (8/ 2) until free F-5-CADA was no longer detectable by thin-layer chromatography (retention factor (Rf) of unbound F-5-CADA ) 0.85). Next, the column was eluted with increasing amounts of methanol in dichloromethane and the fractions were analyzed by thin-layer chromatography using methanol/dichloromethane (1/9). F-5-CADA was detected by a fluorescent lamp (345 nm). Initial fractions containing trace amounts of free F-5-CADA were discarded, and only fractions containing purified labeled block copolymer were (13) Bogdanov, B.; Vidts, A.; Van Den Bulcke, A.; Verbeeck, R.; Schacht, E. Polymer 1998, 39, 1631-1636.

Langmuir, Vol. 22, No. 8, 2006 3571 collected and freeze dried. The retention factors of F-5-CADAPCL-b-PEO and unlabeled polymer were similar (Rf ) 0.5). However, the fluorescence (345 nm) was detected only with F-5-CADA-PCLb-PEO, containing F-5-CADA attached to the polymer. Estimation of the Hydrophobicity of F-5-CADA. Fifty microliters of 200 µM F-5-CADA (in dimethylformamide) was placed into 300 µL of n-octanol, and 300 µL of water was added. The mixture was vortexed vigorously (MaxiMix II M37615, Barnstead Intl., 50 W) at maximum speed for 4 × 20 s, with 10 s pauses in between. The mixture was placed on a belly dancer (Clays Adams Nutator) for 30 min and vortexing was repeated. The phases were allowed to equilibrate at ambient temperature. Aliquots of the n-octanol phase and the water phase were mixed with the same volume of 1 M NaOH to activate the F-5-CADA, and fluorescence was recorded using a FLUOstar OPTIMA (BMG Labtech) spectrofluorometer. The obtained values were corrected for quenching by n-octanol (equimolar activated F-5-CADA measured in water and octanol, water/octanol ) 1.44) and partition coefficient log P calculated as the log of the ratio of corrected fluorescence values in octanol over water (log P ) 1.6). Preparation of Fluorogenic Micelles. PCL-b-PEO polymer was stirred with F-5-CADA-PCL-b-PEO polymer (65/35) in dimethylformamide for 1 h. Next, MilliQ water was added dropwise with continued stirring. Dimethylformamide was removed by dialysis (Spectra/Por membrane, MWCO ) 15 000) against water for a minimum of 24 h with at least eight changes of water. Micelles were sterile-filtered using a 13 mm syringe filter (0.2 µm, Whatman 67861302). The content of F-5-CADA in micelles was determined by spectrofluorometry (SpectraMax Gemini plate reader, excitation at 485 nm, emission at 520 nm). The micelle size was determined by dynamic light scattering using an HPPS particle size analyzer (Malvern Inc.). Integrity Assessment of Micelles in Media. Aliquots of fluorogenic micelles (111 ( 13 µM dye; 1399 ( 136 µM polymer; mean ( standard deviation, n ) 4) were incubated in the medium (phosphate-buffered saline, RPMI 1640, RPMI 1640 with 5% serum, or fetal bovine serum) at a final polymer concentration of 50 µM (4 µM dye) for 0-48 h. Fluorescence corresponding to the spontaneous disruption of the integrity of the micelles was recorded (485 nm, 520 nm) at the end of each time point. The maximal detectable fluorescence was determined by adding dimethylformamide (20% final) to facilitate the breakdown of remaining micelles and NaOH (0.1 M final) to activate F-5-CADA fully. The plate (polypropylene, resistant to DMF and NaOH) was shaken once every 20 min, and the fluorescence was recorded after 1 h. The percent disruption of the integrity of the micelles was calculated from the ratio of the fluorescence at the end of each time point and the maximum detectable fluorescence. Control, equimolar, free F-5-CADA (20 mM stock in dimethylformamide) was treated in the same manner under identical conditions. A FLUOstar OPTIMA (BMG Labtech) spectrofluorometer was used to measure the fluorescence (excitation at 485 nm, emission at 520 nm). Data represent the mean values from at least two independent experiments ( the standard error of the mean. Integrity Assessment of Micelles in Cells. Human urinary bladder carcinoma cells (T24), ATCC number HTB-4, were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2 in McCoy’s 5a medium with L-glutamine, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells (24 well plates, Sarstedt, 83.1836, 105 cells/cm2, 500 µL of serum-free media per well) were incubated in the presence of fluorogenic micelles (concentration response 0-150 µM polymer, 1 h; time response (50 µM polymer) 0-20 h) or control-free dye (0-12 µM F-5-CADA, 1 h; time response (4 µM dye) 0-20 h). F-5-CADA was prepared as a 20 mM stock in DMSO and diluted in culture medium immediately before addition to cells (500 nm emission, 1.5 mm resolution, 0.614 mW laser power, 1 s integration time). Mice were anesthetized (Vetland, Landmark Veterinary Anesthesia System, serial number 81965, Louisville, KY) using a mixture of isofluran (Aerrane, Baxter, CA2L9108) and medical oxygen (Praxair, Montreal) according to McGill University’s standard operating procedure. Micelles (50 µL, 1.4 mM polymer) were administered in 0.9% saline intramuscularly by a single injection in the thigh or subcutaneously below the scruff. Fluorescence was monitored over time (at 0, 1, and 24 h) at the site of injection, and in the body (pelvis, abdomen, and thorax). Each animal was scanned for background before any treatments. The results were displayed using a depth tool option of the default SAMI software. Care was taken to include the identical number of scan points in the analyzed area of each animal. The numerical values of the average concentration index (dimen-

Figure 2. Diameter and fluorescence intensity measurements of flurogenic micelles. The size of the micelles was confirmed by dynamic light scattering (A). Intact fluorogenic micelles were virtually nonfluorescent (B, 6.4 × 101 au; mean plus standard error of the mean). Destroyed micelles (DMF) were intensely fluorescent after chemical activation of fluorogenic F-5-CADA by NaOH (B, 3.6 × 104 au; mean plus standard error of the mean). Data are representative of three independent experiments. **p < 0.001. n1, n2, n3, n4, n5 ) independent samples from different preparations of fluorogenic micelles. sionless measure of fluorescence) were normalized to the signalto-noise ratio of the average background scans of all animals within the group (n ) 4 to 5 per group) and plotted on the y axis. Data Analyses. Data were analyzed by SYSTAT software version 10. Statistical analyses included a student t-test, one way analyses of variance (ANOVA) followed by Tukey’s multiple comparison test, or a t-test with Bonferroni correction as indicated in the text and Figures. P < 0.05 was considered to be significant.

Results Preparation of Fluorogenic PCL-b-PEO Micelles. We have covalently attached F-5-CADA to the PCL part of the PCL21b-PEO45 polymer (Figure 1A). The attachment of F-5-CADA was confirmed by NMR (Figure 1B). The chemical shifts at 2.5 and 3.3 ppm refer to solvents d6-DMSO and DMSO-HOD. The broad chemical shift at 3.56 ppm corresponds to methylene units (g) of the PEO block, whereas chemical shifts at 1.28 (k), 1.57 (i, j), 2.49 (h), and 3.97 (f) correspond to methylene units of the PCL block. The chemical shift at 4.11 (e) ppm refers to the

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Figure 3. Time course of the activation of F-5-CADA in DMF/ NaOH. Fluorescence intensity from both the destroyed fluorogenic micelles (full squares) and control free F-5-CADA (empty diamonds) reached the maximum 60 min after the addition of DMF and NaOH. Data are representative of two independent experiments.

terminal methylene group bearing the carbamate bond, and aromatic chemical shifts Ha, Hb, Hc, and Hd correspond to F-5CADA. The labeled polymer was purified by gel chromatography to remove nonconjugated F-5-CADA (Materials and Methods), and the success of labeling was determined by fluorescence spectroscopy. The labeled polymer was (i) dissolved in dimethylformamide (0.01 mg‚mL-1), (ii) the attached F-5-CADA dye was activated by the addition of NaOH (0.58 M), and (iii) the number of attached F-5-CADA molecules was determined from a linear part of the F-5-CADA’s fluorescence standard curve measured at 485 nm excitation and 520 nm emission. The dye/ polymer ratio obtained in this manner was 0.34, suggesting a 34% success of attachment of F-5-CADA to PCL-b-PEO. (Deactivation of the transitional F-5-CADA-isocynate derivative during the synthesis was a likely contributor to the observed lower value.) Fluorogenic micelles were made from PCL21-b-PEO45 block copolymer and F-5-CADA labeled PCL21-b-PEO45 polymer. The total polymer concentration was 1399 ( 136 µM, and the concentration of F-5-CADA covalently attached to PCL21-bPEO45 was 111 ( 13 µM (mean ( standard deviation, n ) 4), providing micelles with 8% of the labeled polymer. The average diameter of micelles was 21 nm (Figure 2A), which is in agreement with our previous results obtained with tetramethylrhodamine5-carbonyl azide-labeled micelles.14 Intact fluorogenic micelles were virtually nonfluorescent whereas destroyed micelles with subsequently chemically (alkalinization) activated F-5-CADA showed easily detectable fluorescence (Figure 2B). This demonstrates that there was no significant activation of fluorogenic dye during the preparation of micelles. Integrity of Micelles in Media. The percent disruption of micelle integrity was determined from a ratio of fluorescence measured immediately at the end of the experiment and fluorescence intensity 1 h after the addition of DMF/NaOH. DMF was used to destroy the micelles, and NaOH was used to activate the F-5-CADA attached to PCL21-b-PEO45. The equimolar F-5-CADA control was used to monitor the extent of dye activation in various media. Both the fluorogenic micelles and control dye showed easily detectable fluorescence upon addition (14) Luo, L. B.; Tam, J.; Maysinger, D.; Eisenberg, A. Bioconjugate Chem. 2002, 13, 1259-1265.

Figure 4. Integrity of micelles in phosphate-buffered saline. Maintained integrity of fluorogenic micelles in phosphate-buffered saline after 1 h (A), 24 h (B), and 48 (C) h (mean plus standard error of the mean. (A) n ) 6, df5, not significant; (B) n ) 5, df4, p < 0.001; and (C) n ) 3, df2, p < 0.002; t-test, two groups. Data are representative of two independent experiments. F-5-CADA activation (%) ) [(fluorescence of the sample at the end of experiment)/ (fluorescence of the sample after 1 h of incubation in DMF/NaOH added after the measurement of fluorescence at the end of the experiment)] × 100.

of DMF/NaOH (Figure 3). Under the employed conditions, the fluorescence intensity reached the maximal attainable values after 1 h. Therefore, all samples were incubated for 1 h after the addition of DMF and NaOH to ensure the maximum attainable levels of fluorescence. The percent disruption of micelle integrity was calculated as follows: percent disruption of micelle integrity ) [(fluorescence intensity of the sample at the end of the experiment)/(fluorescence intensity of the sample after 1 h of incubation in DMF/NaOH added after the measurement of fluorescence intensity at the end of the experiment)] × 100. The integrity of the micelles was first investigated in phosphatebuffered saline (Figure 4). The fluorescence observed after 2 days of incubation at 37 °C remained below 2% of the maximum (Figure 4A-C). This result demonstrated the maintained micelle integrity in phosphate-buffered saline for 48 h. The fluorescence of control F-5-CADA reached 33% by day 2, demonstrating the partial hydrolytic activation of F-5-CADA in phosphate-buffered saline (Figure 4A-C).

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Figure 6. Integrity of micelles (5, 50, 150 µM polymer) in serum. Disrupted integrity of fluorogenic micelles ranging in polymer concentration from 5 to 150 µM after 24 or 48 h of incubation in fetal bovine serum [mean plus standard error of the mean; 24 h: ANOVA df(2,21), p < 0.05, Tukey’s post-hoc test: 5-50 µM (not significant), 5-150 µM (p < 0.001), 50-150 µM (p < 0.05); 48 h: ANOVA, df(2,21), p < 0.05, Tukey’s post-hoc test; 5-50 µM (not significant), 5-150 µM (p < 0.001), 50-150 µM (p < 0.05)]. Data are representative of two independent experiments. F-5-CADA activation (%) ) [(fluorescence intensity of the sample at the end of experiment)/(fluorescence intensity of the sample after 1 h of incubation in DMF/NaOH added after the measurement of fluorescence intensity at the end of the experiment)] × 100.

Figure 5. Integrity of micelles in RPMI 1640 and fetal bovine serum. Disrupted integrity of fluorogenic micelles in media of increasing biological complexity after 1 h (A), 24 h (B), and 48 h (C) [mean plus standard error of the mean; (A) fluorogenic micelles ANOVA, n ) 16, df(2,13), p < 0.001; free dye ANOVA, n ) 16, df(2,13), p < 0.001; (B) fluorogenic micelles ANOVA, n ) 15, df(2,12), p < 0.001; free dye ANOVA, n ) 16, df(2,13), p < 0.03; and (C) fluorogenic micelles, ANOVA, n ) 10, df(2,7), p < 0.001; free dye, ANOVA, n ) 10, df(2,7), p < 0.02; Tukey’s multicomparison test p < 0.05*, p < 0.001**]. Data are representative of two independent experiments. F-5-CADA activation (%) ) [(fluorescence intensity of the sample at the end of experiment)/(fluorescence intensity of the sample after 1 h of incubation in DMF/NaOH added after the measurement of fluorescence intensity at the end of the experiment)] × 100.

Next, we tested the stability of micelles in cell culture media in the presence or absence of serum. The fluorescence from fluorogenic micelles after 2 days of incubation at 37 °C in serumfree RPMI 1640 was 13% (Figure 5A-C). Compared to phosphate-buffered saline (2%, Figure 4C), these results suggest that the loss of integrity was more pronounced in RPMI 1640. Further increases in the loss of micelle integrity were observed upon the addition of 5% serum to RPMI 1640 (Figure 5A-C). After 48 h, the fluorescence from fluorogenic micelles in the presence of 5% serum in RPMI 1640 reached 64% (Figure 5C). To corroborate the adverse effect of serum on the integrity of PCL-b-PEO micelles, micelles were incubated in 100% serum

(Figure 5A-C). This led to further losses of micelle integrity at later times (74%, Figure 5A and C) and a loss of micelle integrity during the first hour of incubation (59%). The activation of equimolar free F-5-CADA was time- and media-dependent, reaching complete activation in serum after 24 h (Figure 5B). Testing the micelle integrity at 10 times lower (5 µM) and 3 times higher (150 µM) concentrations (compared to 50 µM) showed a significantly lower loss of integrity at 150 µM polymer concentration and no difference at 5 µM polymer concentration (Figure 6). This demonstrates a modest (10-12%) decrease in the loss of micelles integrity at higher polymer concentrations (150 vs 5 µM). Integrity of Micelles in Cells. Next, we investigated the micelle integrity in cells (T24 human bladder carcinoma cells (ATCC, HTB-4)). The micelle integrity was assessed from a ratio of fluorescence detected in cell lysates at the end of each experiment to the maximal attainable fluorescence obtained by full activation of F-5-CADA upon addition of NaOH. The results revealed a time-dependent increase in fluorescence in cell lysates (Figure 7A). Losses of micelle integrity in cells following 0.25, 1, and 20 h of incubation were 9, 14, and 20%. Similar profiles were obtained in the presence of 10% serum (data not shown). The concentration/fluorescence-response study was carried out with 10 and 100 times lower concentrations of micelles in the medium (0.5 and 5 µM). The results revealed an increased loss of micelle integrity in cells (60, 44, and 20%) incubated in the presence of lower starting concentrations of micelles (0.5, 5, and 50 µM) after 20 h of incubation (Figure 7B). Depending on the starting concentration of fluorogenic micelles, the amount of internalized polymer was ∼0.1-7 µg/ million cells. The fluorescence of equimolar F-5-CADA control at the end of each time point was >20 units, and that of the micelles with disrupted integrities was