Poly(amidoamine) Conjugates with Disulfide-Linked Cholesterol

Biomacromolecules 2008, 9, 2693–2704

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Poly(amidoamine) Conjugates with Disulfide-Linked Cholesterol Pendants Self-Assembling into Redox-Sensitive Nanoparticles Elisabetta Ranucci,*,†,‡ Marco A. Suardi,†,‡ Rita Annunziata,† Paolo Ferruti,†,‡ Federica Chiellini,§ and Cristina Bartoli§ Dipartimento di Chimica Organica e Industriale, Universita` di Milano, via Venezian 21, 20133 Milano, Italy, CIMAINA, Centro Interdisciplinare Materiali e Interfacce Nanostrutturate, via Golgi 19, 20133 Milano, Italy, and Laboratorio di Materiali Polimerici Bioattivi per Applicazioni Biomediche ed Ambientali (BIOlab), UdR INSTM, Dipartimento di Chimica e Chimica Industriale, Universita` di Pisa, via Vecchia Livornese 1291, 56122 S. Piero a Grado, Pisa, Italy Received February 4, 2008; Revised Manuscript Received July 31, 2008

Poly(amidoamine) (PAA) networks that are obtained by the use of cystamine as a cross-linking agent in the reaction with 2,2′-dithiodipyridine turn into linear PAAs with dithiopyridyl side groups that easily undergo an exchange reaction with thiocholesterol. The resultant products represent the first examples of amphiphilic PAA-cholesterol conjugates in which lipophilic cholesterol moieties are linked to the hydrophilic PAA chain by S-S bonds that are stable in blood but cleavable inside cells. In aqueous media, these conjugates self-assemble into nanoaggregates whose inner cores consist of lipophilic cholesterol domains. A series of PAA-cholesterol conjugates that are derived from two different bis-acrylamides, namely 2,2-bis(acrylamido)acetic acid and 1,4bis(acryloyl)piperazine, and that have different cholesterol contents were obtained. All products were characterized by 1H and 13C NMR spectroscopy, and the average molecular weights of the soluble polymers were determined by size exclusion chromatography. In all instances, the segregation of cholesterol residues from the aqueous medium was revealed by the comparison of their NMR spectra in CDCl3 and D2O, respectively. The TEM analysis of the PAA-cholesterol aggregates in aqueous buffers revealed homogeneous round nanospheres whose dimensions and dimension distributions were determined by DLS. Preliminary cytocompatibility tests demonstrated that all prepared PAA-cholesterol samples are cytocompatible and thus show potential for biotechnological applications.

Introduction Poly(amidoamine)s (PAAs) are a family of biocompatible and biodegradable polymers that are obtained by Michael-type polyaddition of amines to bisacrylamides.1-4 The first extensive studies on PAA synthesis were published in 1970,5 and afterward, PAA chemical properties and biomedical applications were reviewed in several instances.6,7 Amphoteric PAAs that contain two moderately weak tertamino groups and a single strong carboxyl group per repeating unit and that are predominantly anionic in extracellular fluids8 exhibit stealthlike behavior; that is, after they are intravenously injected in test animals, they are not captured by the reticuloendothelial system but instead have a prolonged permanence in the circulatory system and passively concentrate in tumor tissues, if present, by the so-called EPR (enhanced permeation and retention) effect.9,10 On the contrary, purely cationic PAAs concentrate in the liver, behaving in this respect as poly-L-lysine or polyethylenimine, which, however, are remarkably more toxic.9 Cells internalize both amphoteric and nonamphoteric PAAs via the endocytic pathway. In intracellular compartments, where the pH is lowered first to 6.5 (endosomes) and then to ∼5.5 (lysosomes), PAAs increase their cationic character and may display endosomolytic properties and thus promote the intra* Corresponding author. E-mail: [email protected]. Tel: 0039 02 50614132. Fax: 0039 02 50314129. † Universita` di Milano. ‡ Centro Interdisciplinare Materiali e Interfacce Nanostrutturate. § Universita` di Pisa.

cellular trafficking of biologically active substances, including DNA and proteins.11-15 Smart PAAs that bear disulfide linkages that are regularly arranged along the backbone and are therefore amenable to both hydrolytic and reductive degradation have been reported.16-19 In recent times, we have also reported on the synthesis of linear PAAs with dithiopyridyl side groups that easily undergo exchange reactions with thiol-containing biologically active molecules.20 These PAAs are a step in the synthesis of novel PAA conjugates in which active substituents are bound to the polymer chain through a disulfide linkage that is known to be stable in the bloodstream but amenable to reductive cleavage inside cells.21-26 Broadly speaking, in aqueous media, amphiphilic polymers with hydrophilic and hydrophobic segments may assemble into polymeric micelles or nanoparticles, where the hydrophobic segments segregate from the aqueous environment to form lipophilic domains surrounded by hydrophilic domains. These nanoaggregates are usually endowed with a high drug-loading capacity of the lipophilic cores, and their surfaces may be engineered so as to exhibit biocompatibility that is associated with optimal body distribution. Because of these unique properties, polymeric micelles or nanoparticles have been widely investigated as carriers of bioactive compounds for specific intracytoplasmic delivery or as probes for in vivo imaging applications.26-28 Cholesterol is one of the most common membrane sterols, and it plays a relevant role in the selfassociation of molecules in biological environments. Cholesterol conjugates of hydrophilic polymers such as polysaccharides exhibit a strong tendency toward self-association even at low

10.1021/bm800655s CCC: $40.75  2008 American Chemical Society Published on Web 09/10/2008

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Biomacromolecules, Vol. 9, No. 10, 2008

cholesterol contents 29,30 and have been studied as gene transfection agents, promoters of intracellular delivery of proteins, carriers of lipophilic anticancer drugs, and scaffolds for tissue engineering.31-41 On the basis of this premise, we thought it would be interesting to report here on the preparation, the NMR and morphological characterization, and the preliminary cytotoxicity evaluation of new PAA-cholesterol conjugates in which the cholesterol pendants are linked to the polymer backbone through redox-sensitive disulfide bonds. In aqueous media, these PAA-cholesterol conjugates are expected to self-assemble into hydrophilic/hydrophobic nanoaggregates or nanogels that are characterized by strong hydration of the outer shell because of the highly hydrophilic nature of the PAA portion and are endowed with a significant loading capability of lipophilic drugs in the hydrophobic domains. Because the PAA portion will be exposed to the external medium in biological fluids, these conjugates should behave like the parent PAAs and maintain, inter alia, the ability to be internalized in cells and to promote the intracellular trafficking of bioactive substances. After internalization, however, the relatively high concentration of glutathione that is present in the intracellular environment will cause the cleavage of the disulfide linkages by reduction and trigger the release of a drug payload, if present, by disrupting the nanoaggregates’ architecture.

Ranucci et al. Scheme 1. Synthesis of PAA-SSPy Intermediatesa

Experimental Section Four different PAA-cholesterol conjugates have been prepared: BP-SSChol1 and BP-SSChol2, which are based on 1,4-bis(acryloyl)piperazine (BP), and BAC-SSChol1 and BAC-SSChol2, which are based on 2,2-bis(acrylamido)acetic acid. The reaction pathway consisted of three steps: (1) the synthesis of a PAA-based hydrogel (HG-BP1, HG-BP2, HG-BAC1, and HG-BAC2) containing cystamine as cross-linker (Scheme 1a) in different amounts (number 1 and 2 of the product labels), (2) a disulfide-exchange reaction with 2,2′-dipyridyl disulfide that leads to soluble linear polymers containing ethenyldithiopyridine moieties (BP-SSPy1, BP-SSPy2, BAC-SSPy1, and BAC-SSPy2) (Scheme 1b); and (3) a thiol-exchange reaction between thiocholesterol and the dithiopyridine moieties (BP-SSChol1, BP-SSChol2, BAC-SSChol1, and BAC-SSChol2) (Scheme 2). Instruments and Methods. All NMR experiments were performed on a Bruker Avance 500 spectrometer operating at 500.13 and 125.62 MHz (1H and 13C NMR, respectively). Soluble PAA Conjugate Polymers. The 1D 1H and 13C NMR spectra were recorded with a 5 mm Bruker QNP probe head by the use of standard Bruker software sequences at room temperature in D2O and 95:5 v/v CDCl3/CD3OD as solvent with tetramethylsilane (TMS) as an internal reference. The 13C quantitative spectra were recorded with the inverse-gated decoupled methodology. After several experiments, a 15 s delay was used; longer delay times did not affect the integral measurements. Insoluble PAA Conjugate Polymers. The high-resolution magic angle spin (HR-MAS) 1H and 13C spectra were recorded with a 4 mm Bruker 1H/13C HR-MAS gradient probe at room temperature. The samples were previously swollen in aqueous (5% DCl, D2O) or organic solvent (CDCl3), were packed into a 4 mm HR-MAS rotor (50 µL sample volume), and were spun at 8 kHz at a temperature of 310 K. Two-Dimensional Spectra. All 2D experiments were acquired with the appropriate probe (see above) that worked in inverse detection mode and used z-gradient. The gradient was shaped by a waveform generator and was amplified by a Bruker B-AFPA-10 amplifier. A sinusoidal gradient of 1 ms and a recovery time of 0.1 ms were used. We recorded the 2D COSY, heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond coherence (HMBC) spectra by using the sequences cosygpqf, inv4gpqf, and

a If R1 is -CH2-CH2- and R2 is -CH2-, then BP series polymers are intended. If R1 is -CH(COOH)- and R2 is -H, then BAC series polymers are intended. PySSPy is 2,2′-dipyridyldisulfide.

inv4gplrndqf, respectively. The following acquisition parameters were applied: a 3.3 ms prescan delay and a 5:3:4 gradient combination for HMQC and a 60.0 ms delay for the evolution of long-range coupling (JC,H ) 8.3 Hz) for the HMBC experiment. Size exclusion chromatography (SEC) traces of polymers that are soluble in organic solvents (polystyrene standards) were obtained with Phenomenex Phenogel 500, 103, and 104A columns operating at rt and connected in series, with the UV detector operating at 254 nm, and with a 9:1 (v/v) CHCl3/CH3OH mobile phase. SEC traces of watersoluble polymer (pullulan standards) were obtained by the use of a Waters 515 HPLC pump instrument, Toso-Haas 486 columns operating at rt and using 0.1 M Tris buffer (pH 8.0 ( 0.1) as mobile phase, and a UV detector operating at 230 nm. UV-vis spectra were run on a Perkin-Elmer Lambda EZ210 spectrometer. For all measurements, quartz cells with a 1 cm path length were used. The concentration of 2-mercaptopyridine was determined at 343 nm in 0.1 M Tris buffer (pH 8.0 ( 0.1) with a calibration curve purposely determined (ε ) 8070 mol-1 · cm-1). IR spectra were recorded with a Jasco FT/IR 4100 spectrometer between 400 and 4000 cm-1 with 4 cm-1 spectral resolution; Elemental analyses were conducted with a Fison EA 1108 CHNS apparatus. Specific optical rotations were measured with a Jasco P-1030 polarimeter. Transmission electron microscopy (TEM) analyses were conducted with Zeiss LEO 912AB (EFTEM). One drop of 0.1 mg/mL nanoparticle dispersion was placed on a polymer-coated sample grid after filtration

Poly(amidoamine) Conjugates


Scheme 2. Synthesis of PAA-SSChol Polymersa

HPLC syringe filter. Samples were analyzed with a Viscotek 802 DLS with a detection angle of 90° and a laser wavelength of 830 nm, and data were analyzed with Omnisize 2.0 software with a correlator resolution 256 channel. Materials. Tris(hydroxymethyl)aminomethane (Tris) (>99.8%), 2-mercaptopyridine (>95%), lithium hydroxide monohydrate (>98%), cystamine dihydrochloride (>98%), and reduced glutathione (>97%) were purchased from Fluka and were used as received. Analytical grade HPLC solvents were purchased from Fluka and were used as received. CDCl3 (99.8%) that was stabilized over a silver coil, D2O (99.9%), 2,2′-dipyridyl disulfide (98%), thiocholesterol (98%), and azobenzene (>98%) were purchased from Aldrich and were used as received. 2-Methylpiperazine was purchased from Fluka and was used after recrystallization from n-heptane. The final purity was determined by acidimetric titration. BAC42 and BP43 were synthesized as previously described. Cell line 3T3/BALB-c Clone A31 mouse embryo fibroblasts (CCL163) were obtained from the American Type Culture Collection (ATCC) and were propagated as indicated by the supplier. Dulbecco’s modified Eagle’s medium (DMEM), 0.01 M (pH 7.4) phosphatebuffered saline without Ca2+ and Mg2+ (PBS), calf serum (CS), trypsine/EDTA, glutamine, and antibiotics (penicillin/streptomycin) were purchased from Gibco-BRL. Cell proliferation reagent watersoluble tetrazolim salt (WST-1) was purchased from Roche Diagnostic. Tissue-culture-grade disposable plastics were obtained from Corning Costar. Synthesis of BP-SSPy2. We prepared polymer samples BP-SSPy1, BP-SSPy2, BAC-SSPy1, BAC-SSPy2 by following a previously described procedure.23 As an example, we report in brief the synthesis of BP-SSPy2. In a two-necked flask, BP (1.940 g, 10.0 mmol), cystamine dihydrochloride (0.338 g, 1.5 mmol), and lithium hydroxide monohydrate (0.126 g, 3.0 mmol) were dissolved in water (3.5 mL) under nitrogen flow. 2-Methylpiperazine (0.701 g, 7.0 mmol) was then added under stirring until a homogeneous solution was obtained. Stirring was stopped, and the solution was allowed to react for 120 h. A small portion (20 mg) of the transparent gel obtained (HG-BP2) was conditioned with 1 M aqueous hydrochloric acid, was washed with water and acetone, and was dried under vacuum and stored for NMR analysis. The remaining part of the hydrogel was finely ground and was soaked in a water/ethanol mixture (35 mL of 80:20 or 30:70 v/v for BP and BAC hydrogels, respectively), and 2,2′-dipyridyl disulfide (0.990 g,

a If R1 is -CH2-CH2- and R2 is -CH2-, then BP series polymers are intended; if R1 is -CH(COOH)- and R2 is -H, then BAC series polymers are intended. * In BP-SSChol syntheses, a 9:1 methanol/chloroform mixture was used as solvent. In BAC-SSChol syntheses, a 1:1 water/chloroform emulsion was used as reaction medium.

on a 0.45 µm HPLC syringe filter. The excess was dried out, and the sample was stained with uranyl acetate. Particle size distributions were measured by the use of a Viscotek 802 dynamic light scattering analyzer. We prepared samples by dispersing 1 mg of lyophilized nanoparticles in 1 mL of distilled water in an ultrasonic bath for 15 min. We then diluted the mixture with a 3-fold amount of water or a 10 mM phosphate buffer solution and, if necessary, adjusted the pH by adding a few drops of a 0.1 M HCl or NaOH solution to reach pH 3 or 7.4, respectively. We eventually filtered the mixture on a 1 µm Table 1. Relevant 1H and

HG-BP

1

H

HG-BAC

1′b

2′b

Me-1

3

2.91

2.32; 305 57.5

2.72; 3.25 49.7

2.55; 3.15 51.7

1.19

2.87

2.74

15.2

52.65

172.1

3.00; 3.55 55.9

3.25; 3.66 49.5

3.15; 3.55 50.3

1.39

3.30

28.0 29.15 2.76

15.0

53.6

30.4 31.3

172.6

55.15 3.55

H

13

C

C Chemical Shifts (δ) of Hydrogel Samplesa

2b

1

C

13

1

13

57.4

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4/9

5/8

6

7

3.65

3.65

44.9 45.2 5.57

41.4 41.7

59.3

174.05

10b

11

12

13

14

2.92; 3.32 48.2

3.42

3.04

3.32

2.74

45.8

32.6

43.7

29.55

3.30; 3.66 49.5

3.55

2.85

3.45

2.77

51.0

30.0

50.3

31.3

a HG-BAC hydrochloride was swollen in D2O, and HG-BP was swollen in D2O/DCl (95/5). For m/n ratio, see the text. system.

b

For 1H resonance: AB

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Table 2. Relevant 1H and

a

Ranucci et al.

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C Chemical Shifts (δ) of BP-SSPy and BAC-SSPy Hydrochlorides in D2Ob

For 1H resonance: AB system.

b

For m/n ratio, see the text.

4.5 mmol) and a few milligrams of 2-mercaptopyridine were added. The reaction mixture completely dissolved in a few hours. It was stirred at room temperature for an additional 24 h, and it was then diluted to 100 mL with water to precipitate most of the excess 2,2′-dipyridyl disulfide, which was filtered off. After the pH was adjusted to 2.5 by the addition of 1 M aqueous HCl, the final solution was ultrafiltered through a membrane with a nominal MW cut off of 3000 and was eventually freeze dried. Polymers of the BP series, as hydrochlorides, were soluble in water, methanol, dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF) but were insoluble in chloroform, acetone, toluene, ether, and ethyl acetate. As free bases, they were soluble in DMSO, DMF, chloroform, and methanol but were insoluble in water, acetone, toluene, ether, and ethyl acetate. Polymers of the BAC series, both as hydrochlorides and free bases, were soluble in water and methanol but were insoluble in DMSO, DMF, chloroform, acetone, toluene, ether, and ethyl acetate. j n ) 40 000, M j w ) 130 000, M j w/M j n ) 3.2 (organic BP-SSPY1: M eluent); yield ) 83.4%; yield of the exchange reaction ) 77.5%. j n ) 9500, M j w ) 32 000, M j w/M j nj ) 3.3 (organic eluent); BP-SSPY2: M yield ) 70.4%; yield of the exchange reaction (from NMR data) ) j n) 26 000, M j w) 52 000, M j w/M j n ) 2 (aqueous 75.4%. BAC-SSPY1: M eluent); yield ) 85.6%; yield of the exchange reaction ) 73.1%. j n) 44 000, M j w ) 93 000, M j w/M j n ) 2.1 (aqueous BAC-SSPY2: M eluent); yield ) 68%; yield of the exchange reaction ) 73.6%. In Tables 1 and 2, the full NMR characterization of the products is reported. The 13C NMR quantitative spectrum allowed for the determination of the ratio between the different repeating units. Therefore, the integration of all C-6/C-7 versus C-Me-1 signals was performed in the case of HG-BP and BP-SSPy, respectively, and the integration of all C-6 versus C-Me-1 was performed in the case of HG-BAC and BAC-SSPy, respectively. These measurements showed a n/m ratio of 80:20 and 81:19 for HG-BP2 and HG-BAC2, respectively. The cross-linking degree of the HG-BAC hydrogel, which was calculated by the integration of C-12 (CH2-S-S carbon at 30.0 ppm) versus all C-4 and C-9 (CH2-CO carbons at 28.0 and 29.15 ppm, respectively), confirmed the above results. The n/m ratios of the pyridyl derivatives, which were recovered after the exchange reaction, were 84:16 and 86: 14 for BP-SSPy2 and BAC-SSPy2, with an exchange reaction yield of >70% in both cases.

Figure 1. Structure of vinyl terminus present in HG-BP2 product.

In the case of HG-BP samples, the 1H and 13C spectra revealed the presence of a limited amount of vinyl functions. These must correspond to dandling chain ends (Figure 1), which are easily recognizable for their higher mobility with respect to the backbone units. The number of vinyl dandling terminals, which was determined from the quantitative 13C spectra, was about 2.3% of the overall number of 1,4-BP-deriving units. Synthesis of BP-SSChol2. BP-SSPy2 hydrochloride (1.005 g, 2.6 mmol repeating units, 0.43 mmol SSPy units) and thiocholesterol (0.191 g, 0.47 mmol) were introduced in a two-necked flask that was equipped with magnetic stirring and were purged with nitrogen. Deoxygenated chloroform/methanol (9:1, 60 mL) and triethylamine (0.3 mL, 2.6 mmol) were added, and the solids were allowed to dissolve under nitrogen. The solution turned yellow in few minutes, and the reaction mixture was allowed to react for an additional 15 h. Then, the organic solution was poured in water (50 mL). The solution pH was adjusted to 2.5 by the addition of 1 M HCl aqueous solution, and an emulsion was obtained. The suspension was concentrated to 40 mL by evaporation in vacuo, and water (50 mL) was then added. The final aqueous suspension was extracted with diethylether (2 × 200 mL) and was then dialyzed with a 12 000 nominal cutoff tube against an 80:20 v/v water/ methanol mixture until the complete disappearance of mercaptopyridine (UV monitored). The ultrafiltered suspension was eventually lyophilized. We prepared BP-SSChol1 by following the previously described procedure. We prepared BAC-SSChol1 and BAC-SSChol2 by following the same procedure but by using a 1:1 water/chloroform emulsion as the reaction medium in place of the methanol/chloroform mixture. BP-SSChol1. Yield ) 60%. Cholesterol content: 7.8% w/w (13C NMR data). Anal. Calcd: C, 58.4; H, 8.0; N, 14.5; S, 1.3. Found: C, 52.3; H, 10.1; N, 14.9; S, 1.35. IR (cm-1): 3422 (amide N-H stretching), 2928-2858 (C-H stretching), 1637 (broad, amide CdO stretching), 1439-1385 (CH2 bending), 1221 (amine C-N stretching), -1 -1 1016 (CH bending). [R]25 · g · cm3 (for comparison, D ) -1.4 deg · dm



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Figure 2. 1H spectra of BP-SSChol2 in (a) CDCl3 and (b) D2O (x-axis unit: ppm). 25 thiocholesterol [R]D ) -23 deg · dm-1 · g-1 · cm3). Solubility as a free base: soluble in chloroform, dichloromethane, methanol, and dimethylsulfoxide and insoluble in water. Solubility as a hydrochloride salt: insoluble in organic solvents; formed suspensions in aqueous solution. BP-SSChol2. Yield ) 62%. Cholesterol content: 16.4% w/w (13C NMR data). Anal. Calcd: C, 62.2; H, 7.9; N, 12.1; S, 2.6. Found: C, 25 51.7; H, 9.4; N, 13.4; S, 3.16. [R]D ) -2.7 deg · dm-1 · g-1 · cm3. Solubility as BP-SSChol1. BAC-SSChol1. Yield ) 61%. Cholesterol content: 7.3% w/w. Anal. Calcd: C, 48.2; H, 7.1; N, 15.7; S, 0.9. Found: C, 50.6; H, 8.2; N, 16.7; S, 0.9. IR (cm-1): 3416 (amide NH stretching), 3281 (carboxylic acid OH stretching), 2967-2828 (CH stretching), 1650-1621 (broad, amide and carboxylic CdO stretching), 1519 (amide NH bending), 1454-1385 (CH2 bending), 1129 (amine C-N stretching), 1134 (CH bending). Solubility: insoluble in water and in organic solvents; formed suspensions in aqueous solution. BAC-SSChol2. Yield ) 62%. Cholesterol content: 13.1% w/w. Anal. Calcd: C, 50.2; H, 7.3; N, 14.3; S, 2.0. Found: C, 50.7; H, 8.2; N, 17.1; S, 2.2. Solubility as BAC-SSChol1. Loading Capacity. We measured the uptake ability of hydrophobic substances by measuring the amount of azobenzene and estradiol that was dissolved in the hydrophobic core of the PAA-SSChol nanoparticle samples. The hydrophobic substance (1.5 mg) was added to the polymer solution (1.5 mg polymer in 1.5 mL of water) and was adjusted to pH 7.4 in 0.01 N PBS in the presence of a small amount of methanol (15 µL). After 48 h, the volume was first reduced under vacuum to remove methanol and was then restored to 1.5 mL with water, and the suspension was filtered with a 0.45 µm pore size filter to remove the undissolved hydrophobic molecule. Methanol was added to the filtered

solution in an equal volume, and absorption intensity was measured by UV-vis spectroscopy at 430 nm for azobenzene and at 280 nm for estradiol. A similar sample without polymer was prepared as a blank, and the hydrophobic molecule absorption was subtracted from the polymer sample absorptions. The amount of hydrophobic substance was determined by a preprepared analytical curve. Stability to Reducing Agents. BP-SSChol1 and BAC-SSChol2 were dispersed in deoxygenated 10 mM PBS, and pH was adjusted to 7.4 with a 0.1 M NaOH solution to obtain a final concentration of polymer of 3.5 mg/mL. Cloudy suspensions were invariably obtained. A 2.5 mL aliquot of a 1% reduced glutathione solution that was previously prepared in deoxygenated water was added to a 3 mL aliquot of the polymer suspensions. The obtained mixtures were incubated for 30 min under stirring until they turned clear. Several 0.5 mL aliquots of reduced glutathione solution were subsequently added, and the polymer solution was incubated for 30 min at 37 °C. The particle diameter was measured with a submicron particle analyzer (Coulter MD, Hialeah, FL) after each single addition/incubation step. We repeated the same procedure by substituting doubly distilled water for reduced glutathione solution, and we used the results as a blank. Biological Evaluations. Cell Line. We carried out cytotoxicity evaluations of the investigated materials by using the 3T3/BALB-c Clone A31 cell line. Cells were grown in DMEM containing 10% CS, 4 mM glutamine, and 100 U/mL:100 µg/mL penicillin/streptomycin (complete DMEM). Subculturing. A 25 mL flask that contained exponentially growing 3T3 cells was observed under an inverted microscope for cell confluence. The complete DMEM media was then removed, and cells were rinsed for a few min with PBS. The buffer solution was removed,

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Table 3. Relevant 1H and

Ranucci et al.

13

C Chemical Shifts (δ) of PAA-Cholesterol Conjugatesb

a BP-SSChol in CDCl3/CD3OD (95:5) as solvent (HR NMR). BAC-SSChol hydrochloride in D2O (HR NMR). BAC-SSChol swelled in CDCl3 (HRMAS NMR). See the text for major explications. b For 1H resonance: AB system.

and cells were incubated with 0.5 mL of trypsin/EDTA solution at 37 °C in a 5% CO2 incubator for 5 min or until the monolayer started to detach from the flask. Cells were suspended in an appropriate volume of DMEM and were plated at a split ratio of 1:6 or 1:10 in a 75 mL flask. For routine culturing and evaluation of morphology, cells were analyzed under a Nikon Eclipse TE2000-U inverted microscope. Determination of IC50 of Polymeric Materials. We evaluated the IC50 (50% inhibitory concentration, that is, the material concentration at which 50% of cell death with respect to the control is observed) of the investigated polymers by exposing cells to DMEM containing different polymer concentrations (1-10 mg/mL) for 24 h. At the end of the exposure time, cells were incubated with the WST-1 cell proliferation reagent for the quantitative evaluation of cell proliferation. Cell Proliferation Assay. We assayed quantitative proliferation by using the cell proliferation reagent WST-1 and by following the protocol that was indicated by the manufacturer. Briefly, cells were allowed to proliferate in DMEM that contained different concentration of polymeric materials, and they were then incubated for 4 h with an appropriate volume of WST-1 tetrazolium salts. Formazan production was detected at 450 nm (620 nm as the reference wavelength) by the use of an ELISA microplate reader (Biorad).

Results and Discussion The new PAA-cholesterol conjugates were obtained by the thiol-exchange reaction of thiocholesterol with PAA precursors that bear ethenyldithiopyridyl pendants (PAA-SSPy). Four PAAs were chosen as precursors: two purely cationic PAAs (BP-SSPy1 and BP-SSPy2) and two amphoteric PAAs (BAC-SSPy1 and BAC-SSPy2). We prepared them by employing BP and BAC, respectively, as bis-acrylamide monomers. Synthesis of PAA-SSPy. A two-step synthetic strategy was adopted (Scheme 1). The first step consisted of preparing PAA-

cross-linked networks by using cystamine as a structure-forming comonomer. Cystamine, in fact, behaves as a tetrafunctional monomer in Michael-type polyadditions to bis-acrylamides, as does any other R, ω primary diamine.6 The second step consisted of a double-exchange reaction of the cystamine units with excess 2,2′-dithiopyridine in the presence of a catalytic amount of 2-mercaptopyridine, which finally gave linear PAA-SSPy. Four different cross-linked PAA samples were obtained by reacting BP (HG-BP1 and HG-BP2) or BAC (HG-BAC1 and HG-BAC2) with mixtures of 2-methylpiperazine and cystamine in different proportions. More precisely, in HG-BP1 and HG-BAC1, the amount of the amine hydrogens of cystamine over the total number of amine hydrogens was 10%, whereas in HG-BP2 and HG-BAC2, this amount was 20%. The exchange reactions were performed on finely ground PAA networks that were swollen in ethanol (HG-BP series) or in a 2:1 (v/v) water/ethanol mixture (HG-BAC series) under conditions that were slightly alkalic (pH of 8.5 to 9 after dilution with water). A 3:1 2,2′-dipyridyl disulfide/cystamine moieties ratio was employed in all cases. The reaction mixture became a clear, homogeneous solution in a few hours, and the dissolution time largely depended on the particle size of the hydrogels. The reaction was then allowed to proceed for an additional 24 h. The final products were purified by ultrafiltration and were recovered by lyophilization. Yields were in the range of 65-86%. Molecular weights and molecular weight distributions were determined by SEC. Different conditions were adopted depending on the solubility properties of the different polymers. In particular, we analyzed polymers of the BP series by using 9:1 (v/v) CHCl3/CH3OH as a mobile phase, whereas we analyzed polymers of the BAC series by using 0.1 M Tris buffer (pH 8.0 ( 0.1) as a mobile phase. Although the molecular weight values were of the same order as those of most of the PAAs that were described so far,5-9 a different trend was


Figure 3.


2699

13

C spectra of BP-SSChol2 in (a) CDCl3 and (b) D2O (x-axis unit: ppm).

observed for the two series. Because the disulfide/disulfideexchange reaction of the HG hydrogels went to completion in all cases, on the basis of the results of the 13C NMR characterizations, these differences in molecular weight cannot be ascribed to the extent of the exchange reaction. In contrast, observed differences can be ascribed to the fact that SEC analyses of BAC and BP polymers were conducted under different experimental conditions, such as stationary phase, eluent, and type of detector. Synthesis of PAA-SSChol. Different PAA-cholesterol conjugates were obtained by a thiol-exchange reaction of thiocholesterol with the ethenyl dithiopyridine units of BP-SSPy and BAC-SSPy precursors (Scheme 2). In the case of BP-SSPy polymers, the thiol-exchange reaction was performed in 1:9 (v/v) methanol/chloroform in which both reactants were soluble, whereas in the case of BAC-SSPy the reaction was performed in a chloroform/water slurry; thiocholesterol resided in the former solvent and BAC-SSPy resided in the latter solvent. The thiol-exchange reactions produced high yields in the presence of triethylamine as the basic catalyst. Stronger inorganic bases invariably lead to undesired side reactions and were therefore discarded as catalysts. When the reaction was complete, water was added to the reaction mixtures, and the raw products were acidified to pH 3.5 by dilute hydrochloric acid. The organic solvents were then stripped under vacuum, and the resultant suspension was extracted with diethyl ether to eliminate excess thiocholesterol and was eventually dialyzed against an 80:20 water/methanol mixture until the 2-mercap-

topyridine byproduct completely disappeared (UV monitoring). The exchange reaction produced about 100% yield, as ascertained by UV spectroscopy, but the dialysis step, which eliminated the low molecular weight fraction, reduced the yield of the final product to 60%. No traces of pyridine groups were detected by NMR in the final products (see below). All products formed stable cloudy suspensions in water. The polymers of the BP series, as hydrochlorides, were insoluble in chloroform, dichloromethane, methanol, and DMSO, whereas, as free bases, they showed good solubility in some organic solvents, such as methanol and chloroform. As a consequence, it was possible to perform specific optical rotation measurements and NMR analyses of these products in solution. Indeed, these products showed specific optical rotation abilities that revealed the presence of chiral moieties linked to the otherwise nonchiral polymer chain. The conjugates of the BAC series were insoluble in organic solvents in all conditions. The IR spectra of all products showed the presence of carbonyl, amino, and amido groups. The absence of peaks due to aromatic compounds confirmed that the reaction proceeded to completeness and that 2-mercaptopyridine was completely eliminated. On the whole, elemental analyses confirmed the expected functionalization degrees of the polymers. Differences in the absolute values are probably ascribable to hydration, but in all cases the proposed structure was confirmed by the good agreement between the found and the calculated nitrogen/sulfur ratio.

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Figure 4. 1H spectra of BAC-SSChol2 in (a) CDCl3 and (b) D2O (x-axis unit: ppm).

NMR Characterization. The structures of BP-SSPy and BAC-SSPy conjugates were first investigated by the combined use of 1D and 2D NMR gradient-enhanced experiments such as 2D COSY, 1H/13C 2D HMQC, and 1H/13C 2D HMBC.44,45 BP-SSPy. The 1H/13C HMQC experiments revealed diastereotopic proton pairs for each CH2 group. They allowed the assignment of the H-6 and H-7 piperazine protons and the H-1, H-2, H-1′, and H-2′ protons of the 2-methylpiperazine rings by the use of the correlations that were found in the COSY spectrum, starting from H-1. Therefore, in the HMBC experiment, the long-range coupling of amidic carbons C-5/C-5′ and C-8/C-8′ with the vicinal methylene protons H-4, H-9, and H-14 allowed us to distinguish the noncorrelated protons H-12 and, consequently, H-11 from the COSY spectrum. (See Table 2 for the assignments). BAC-SSPY. The 2D homo- and heterocorrelated maps revealed the same correlation trends as those previously described for PAA networks19 and as those reported above for BP-SSPy series, which allowed chemical shift assignments (Table 2) for H-12 and H-11. HG-BP and HG-BAC. These compounds, like most crosslinked PAAs, are insoluble in most organic and aqueous solvents but swell in water, giving rise to hydrogels. Among the different analytical methods that are employed in the structural elucidation of insoluble materials, HR-MAS NMR has recently received much attention.46,47 This nondestructive technique, which is at

the borderline between solid-state and solution NMR, was already employed in the characterization of PAA networks.48 We used D2O and 95:5 D2O/DCl as swelling solvents, a 300 K temperature, and a spinning rate of 8 KHz as the best experimental conditions. The complete 1H and 13C assignment was performed and confirmed by a comparison with the spectral data of the soluble series (Table 1). In the case of HG-BP, some differences in the proton resonances were observed with respect to soluble BP-SSPy. This is probably because the hydrogels were analyzed as free bases, whereas the BP-SSPy samples were analyzed as hydrochlorides. BP-SSChol. These compounds, which are soluble in organic solvents, were obtained from the corresponding ethenyldithiopyridyl polymers and were first investigated as free bases in CDCl3. The 1H and 13C chemical shift values of the piperazine and 2-methylpiperazine residues in the macromolecular backbone were in agreement with those previously reported. (See, for instance, Figure 2 and Table 3.) All resonances of the cholesteryl moieties were very similar to those of the thiocholesterol moieties with the exception of those belonging to the A ring, in which the thiol group was replaced by a disulfide function. In particular, the C-15, C-16, and C-20 resonances, which were observed in the 13C spectrum of thiocholesterol at 39.7, 44.2, and 34.0 ppm, respectively, were shifted at 49.9, 38.4, and 28.9 ppm, respectively. These chemical shift changes,


Figure 5.


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13

C spectra of BAC-SSChol2 in (a) CDCl3 and (b) D2O (x-axis unit: ppm).

similar to those observed on passing from thiols to disulfides, suggested the presence of cholesteryl moieties bound to the main backbone with a disulfide linkage. Remarkably, the lines of cholesterol resonances were sharp, whereas those of the PAA backbone were much broader. Line broadening became even more evident in the 13C spectra (Figure 3, panel a). This behavior, which was never observed in the case of the BP-SSPy parents, was ascribed to the tendency of BP-SSChol to undergo phase association. The hypothesis is that in CDCl3, the hydrophilic PAA portion of the conjugates partially segregates to give rise to a reverse micellar structure. This reduces the mobility and gives rise to line broadening. This hypothesis was confirmed by the observation of an analogous, but exactly opposite behavior in D2O in which both proton (Figure 2, panel b) and carbon (Figure 3, panel b) spectra of BP-SSChol hydrochloride revealed only the PAA backbone’s signals and thus indicated the strongly reduced mobility of cholesteryl residues. The obvious explanation is that BP-SSChol conjugates undergo phase segregation of lipophilic cholesteryl domains whereas hydrophilic PAA segments remain well solvated in D2O. BAC-SSChol. Because these conjugates are not soluble in organic solvents but swell in only CDCl3, the HRMAS technique was used. Interestingly, the resulting NMR spectra (Figures 4 and 5) exclusively showed signals that were ascribable to the cholesterol residues and the ethenyldithionyl bridges (Table 3), which was in agreement with the values that were previously found for the cholesteryl moiety of BP-SSChol. In addition, it was possible to recognize the C-11 and C-12 signals of the ethenyldithionyl segment at 44.2 and 29.7 ppm, respectively. These 13C resonances were in agreement with the values that were found in the free-base HG-BP hydrogels (see Table 1 and Figure 5, panel a).

In contrast, the spectra of BAC-SSChol hydrochloride conjugates, which give stable dispersions in D2O, revealed only the PAA backbone’s resonances, which was in agreement with the previously reported BAC-SSPy’s assignments (Figure 4, panel b and Figure 5, panel b). Therefore, the chemical shifts of Table 3 are referred to the spectra of BAC-SSChol swelled in CDCl3 (HRMAS) for the cholesterol residue and to BAC-SSChol hydrochloride in D2O, for the polymer backbone. This result points to the conclusion that in the case of BAC-SSChol conjugates hydrophobic cholesteryl units segregate from D2O into insoluble lipophilic domains and are therefore not revealed by NMR spectroscopy. Morphological Characterization. The dimensions of PAAcholesterol nanoaggregates in aqueous media were determined by DLS, and their morphological analyses were performed by TEM. Each sample was routinely filtered through 1 and 0.45 µm porosity filters prior to DLS and TEM analyses. Filtrate solutions invariably showed, upon drying, a nearly quantitative recovery of the initial sample. Because PAAs carry ionizable groups, DLS analyses were performed at two different pHs, namely pH 3 and 7.4, to evaluate pH-mediated conformational changes. Obtained data, averaged over measurements performed in triplicate, are reported in Table 4. All PAA-cholesterol conjugates were found to selfassemble in nanoparticles with narrow monomodal diameter distributions ranging from 100 to 300 nm. It may be observed that BP-based nanoparticles exhibited invariably larger dimensions than did BAC-based nanoparticles with similar cholesterol content, which is probably due to the lower hydrophilicity of the BP-based PAA backbone, which reduces the dispersion ability and leads to larger aggregates. Moreover, BP-based

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Table 4. DLS Analyses of PAA-SSChol Conjugates pH 3 sample BP-SSChol1 BP-SSChol2 BAC-SSChol1 BAC-SSChol2 a

a

pH 7.4 b

D (nm) 164 ( 3 191 ( 7 112 ( 3 109 ( 4

PI 0.07 0.03 0.05 0.04

D: average nanoparticle diameter.

b

D (nm) 243 ( 16 264 ( 21 124 ( 6 131 ( 7

PI 0.20 0.18 0.11 0.13

PI: polydispersity index.

Figure 7. Stability to reduction assay. Decrease in BP-SSChol1 nanoparticle diameter with increasing concentration of reduced glutathione in the suspension: 0.30, 0.36, 0.41, 0.46, and 0.50% (w/ v).

determined by TEM in the dry state were approximately 3 times smaller than those obtained by DLS in aqueous media. A similar behavior has already been reported for different hydrophilic polymer-based nanoparticles.49,50 This finding confirms the large water absorption ability of PAA-cholesterol conjugates. Therefore, they are true nanohydrogels that are characterized by a water-absorbing PAA matrix and insoluble cholesteryl domains as physical cross-links. Evaluation of Uptake Ability of PAA-Cholesterol Conjugates toward Hydrophobic Substances. We measured the uptake ability of PAA-cholesterol toward two different hydrophobic probes, namely azobenzene and estradiol, by measuring the amount of probe that was solubilized by the conjugates by means of UV measurements in comparison with the amount that was solubilized by plain water and aqueous PAA solutions. Azobenzene was chosen because of its high lipophilicity and its strong absorbance of UV light that make it easy to detect its concentration, even in very dilute solutions. Estradiol was instead chosen for its structure affinity for cholesterol, which was expected to increase the uptake ability. The amount of azobenzeneandestradiolthatwastakenupbyeachPAA-cholesterol sample is reported in Table 5. The uptake abilities were in the range of 9-20% polymer conjugates on a weight-to-weight basis. It may be observed that a good proportional relationship exists between the amount of hydrophobic probes that were solubilized and the amount of cholesterol moieties in the conjugate. This gives a clear indication that lipophilic probes were absorbed in the hydrophobic cholesterol domains. Moreover, the observed uptake ability of the PAA-cholesterol conjugates was comparable to that reported elsewhere with other amphiphilic polymers, even with different lipophilic probes.33-36 Stability to Reducing Agents. The redox sensitivity of PAA-cholesterol nanoparticles was demonstrated by degradation tests that were carried out in the presence of reduced glutathione as the model reducing agent. The decrease in the dimensions of the nanoparticles with increasing glutathione concentration was monitored by LLS

Figure 6. TEM image of BAC-SSChol2 nanoparticles obtained from a pH 3.0 buffer suspension with negative staining technique.

nanoparticles undergo a considerable increase in diameter by increasing pH. This result may be ascribed to nanoparticle aggregation because of the decrease in the net positive charge of BP-based PAAs and hence of the hydrophilicity and ionic chain-to-chain repulsion, which occur at higher pH values. In contrast, BAC-based nanoparticles exhibit only a modest increase in diameter with pH. It may be observed that at all pHs the PAA backbone of BAC-based conjugates is endowed with a higher solubility than the BP-based conjugates, which is due to the presence of a carboxyl function that is always negatively charged at a pH of >3.8 This leads to a better dispersability at all pHs and renders the effect of changes in the amount of positive charge less relevant in this respect. TEM imaging experiments, which were performed on dried samples that were prepared by means of a negative staining technique, invariably lead to results that were similar to those reported in Figure 6, which referred to a BAC-SSChol2 sample. Homogeneous round nanoparticles were revealed and were characterized by a quite regular diameter distribution that was centered around 40 nm. As expected, the nanoparticle diameters

Table 5. Evaluation of PAA-SSChol Conjugates’ Ability to Uptake Hydrophobic Probes sample BAC-SSChol1 BAC-SSChol2 BP-SSChol1 BP-SSChol2 a

azobenzene/PAA-SSChol (mg/g) 132 285 94 210

( ( ( (

7 12 6 8

azobenzene/cholesterol (w/w)a 1.82 1.78 1.09 1.30

( ( ( (

0.10 0.08 0.07 0.05

estradiol/PAA-SSChol (mg/g) 90 124 129 189

( ( ( (

Weight ratio of the lipophilic probe versus the amount of cholesterol contained in the polymer conjugate.

12 7 7 10

estradiol/cholesterol (w/w)a 1.33 0.80 1.48 1.08

( ( ( (

0.18 0.04 0.08 0.06


measurements. The rate of dissolution of small nanoparticles (400 nm diameter) was too high to allow for meaningful measurements. Therefore, to obtain more evident degradation trends, we used relatively high concentrations (3.5 mg/mL) of purposely prepared larger nanoparticles with broad size distributions. In the presence of increasing amounts of reduced glutathione, a gradual diameter reduction was observed, as reported in Figure 7 for BP-SSChol1. Water that was added in the place of reduced glutathione was completely ineffective and thus demonstrates that the diameter reduction was not due to dilution but to only glutathione-induced reduction. The capability of the SS bond to be cleaved by reducing agents, in particular by reduced glutathione, which is the main reducing agent inside cells, represents a very important feature in view of utilizing PAA-SSChol for the intracellular delivery of drugs and bioactive molecules. Because of the redox sensitivity of the SS bond, the PAA-SSChol nanoparticles are expected to be disrupted and to release their drug payload after internalization in cells. Biological Evaluations. The cytotoxicity of PAA-cholesterol conjugates was assessed by in vitro cytotoxicity assays that were performed against 3T3/BALB-c Clone A31 cell lines. Cells were incubated for 24 h with the polymer solutions and were then analyzed for viability by WST-1 tetrazolium salt, which allows for the quantitative evaluation of metabolically active cells. The resulting IC50 values, namely 1.5 ( 0.17, 3.0 ( 0.95, >10.0 ( 2.2, and 3.0 ( 0.87 mg/mL for BP-SSChol1, BP-SSChol2, BAC-SSChol1, and BAC-SSChol2, respectively, point to the conclusion that all PAA-cholesterol conjugates exhibit, on the whole, low cytotoxicity, especially in the case of BAC-SSChol1 (IC50 > 10 mg/mL). Apparently, the cholesterol content does not have the same effect for both polymer series because higher contents decrease the cytotoxicity of BP-based conjugates but increase that of the BAC-based conjugates. However, it is not possible to draw straightforward conclusions regarding the effect of cholesterol content on cytotoxicity because it affects the state of the polymers in aqueous systems, and it was not possible to screen out this factor.

Conclusions Novel stimuli-responsive PAA-cholesterol conjugates have been studied. Because of the presence of cholesteryl pendants, these conjugates form nanosuspensions in aqueous media. Dispersed nanoparticles exhibit a loading ability toward lipophilic molecules and retain the low cytotoxicity profile of PAAs. The most remarkable feature of the new systems consists of the presence of disulfide bonds between the hydrophilic PAA backbone and the lipophilic cholesteryl pendants, which gives rise to the redox sensitivity of nanoparticles that are stable in storage but rapidly disintegrate in the presence of reducing agents. In conclusion, the novel PAA-cholesterol conjugates that are reported in this article can be expected to act as smart systems for the intracellular delivery of lipophilic molecules because of the known ability of PAAs to promote intracellular trafficking and because of the redox-sensitive disulfide bonds between the PAA chains and the cholesterol pendants. Acknowledgment. We acknowledge the financial contribution from the Consorzio Nazionale Scienza e Tecnologia dei Materiali (INSTM) Prisma 2005 project. We also thank Dr. Nadia Santo for performing TEM analyses.

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BM800655S

Poly(amidoamine) Conjugates with Disulfide-Linked Cholesterol

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