Antimitogenic Polymer Drugs Based on AMPS: Monomer Distribution

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Biomacromolecules 2010, 11, 626–634

Antimitogenic Polymer Drugs Based on AMPS: Monomer Distribution-Bioactivity Relationship of Water-Soluble Macromolecules Luis Garcıá-Fernańdez,†,‡ Marıá R. Aguilar,*,†,‡ Marıá M. Fernańdez,§ Rosa M. Lozano,| Guillermo Gimeńez,| and Julio San Romań†,‡ Biomaterials Department, Institute of Polymer Science and Technology (ICTP, CSIC), Juan de la Cierva 3, 28006 Madrid, Spain, Networking Biomedical Research Centre in Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN, Spain, Pharmacology Department, Faculty of Pharmacy, Universidad Complutense de Madrid (UCM), Ciudad Universitaria s/n, 28040 Madrid, Spain, and Department of Protein Function and Structure, Centre of Biological Investigations (CIB, CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain Received October 21, 2009; Revised Manuscript Received December 22, 2009

A number of polysulfonated molecules have demonstrated their interaction with fibroblast growth factor (FGF), hampering their binding to its receptors (low affinity heparan sulfate proteoglycans (HSPG) and high affinity tyrosine kinase FGF receptors) and inhibiting the intracellular signaling and mitogenic response in cultured endothelial cells. The aim of this work was the synthesis and characterization of new copolymers based on 2-acrylamido-2-methylpropane sulfonic acid (AMPS) with antiproliferative activity for antitumoral applications. N-Vinylpyrrolidone (VP) or butyl acrylate (BA) was copolymerized with the sulfonated monomer to obtain macromolecules with different hydrophilic/hydrophobic balance and distribution of the sulfonated groups within the macromolecules. In vitro cell culture proliferative assays showed that monomer distribution affected the inhibition of the proliferative action of FGF. Reactivity ratios of the systems were determined following the free radical copolymerization by in situ 1H NMR, and the correlation of the monomer sequence distribution with the bioactivity is discussed.

1. Introduction Vascular supply is vital for living tissues, both normal and neoplastic, because it guarantees the feeding of the nutrients supply and the clearance of waste products to the cells. Angiogenesis is the natural process that aids in blood vessel proliferation in the human body, especially in neonates and wound healing. Several diseases have been associated with persistent upregulated angiogenesis, such as cancer, diabetes, blindness, and psoriasis. This process is crucial in the development, progression, and metastasis of malignant tumors. Generally, a solid tumor expands until 1-2 mm3 is reached. At this point, metabolic demand increases, and the balance of pro- and antiangiogenic factors changes in favor of the pro-angiogenic molecules. The tumor switches to an angiogenic phenotype and recruits blood vessels from the surrounding tissue, developing a dense vasculature that provides nutrients to the cancerous tissue. Therefore, the control of angiogenesis is a potential therapeutic target for the control and treatment of a wide range of neoplastic pathologies, because the inhibition of angiogenesis in tumors inhibited tumor growth, decreased tumor mass, and induced tumor regression.1–4 Antiangiogenic therapy, which targets activated endothelial cells, presents several advantages if compared to the therapy directed against tumor cells. First, * To whom correspondence should be addressed. Tel.: +34915618806 (ext. 212). Fax: +34915644853. E-mail: [email protected]. † ICTP, CSIC. ‡ CIBER-BBN. § UCM. | CIB, CSIC.

activated endothelial cells are genetically stable and spontaneous mutations rarely occur, which minimizes the probability of these cells to become resistant to the antiangiogenic drugs. Second, turnover of endothelial cells in tumoral tissues may be 50 times higher than that of endothelium in normal tissues, presenting a faster and more active metabolism. Activated endothelium overexpress specific markers that could be used to develop specific therapies directed to these cells. Moreover, activated endothelial cells are easily accessible by systemic administration as they are directly in contact with blood. Finally, different tumor cells are sustained by a single capillary, and consequently, inhibition of a small number of tumor vessels may affect the growth of many tumor cells. Inhibition of angiogenesis can be achieved through several means that include inhibition of the action of angiogenic factors, increase of the production of inhibitors, or inhibition of receptor activation and signaling in blood vessels. Fibroblast growth factors (FGFs) are a very important family of pro-angiogenic polypeptides that spans a large spectrum of activities, such as proliferation, migration, and differentiation of vascular cells. Also, it has been demonstrated that FGFs play a decisive role in tumor angiogenesis.5 Two classes of receptors for FGF have been identified. One comprises a family of transmembrane signaling receptors (FGFRs) with intrinsic tyrosine kinase activity. These bind to FGF with high affinity and are responsible for initiating the observed biological response. The second class of receptor includes a family of cell surface heparan sulfate proeoglycans (HSPG) that bind FGF with low affinity but high capacity and that do not transmit a biological response. It has been reported that FGF binding to these cell surface heparin-like molecules

10.1021/bm901194e  2010 American Chemical Society Published on Web 02/12/2010

Antimitogenic Polymer Drugs Based on AMPS

Biomacromolecules, Vol. 11, No. 3, 2010

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Table 1. Composition and Thermal Properties of the Synthesized Poly(BA-co-AMPS) Copolymers Obtained by 1H NMR, DSC, and TGA composition

poly(BA-co-AMPS) poly(BA-co-AMPS) poly(BA-co-AMPS) poly(BA-co-AMPS)

(25:75) (50:50) (65:35) (75:25)

DSC

F(BA)feed

f(BA)copol

w(BA)copol

yield

25 50 65 75

23 50 64 72

15 38 52 61

86.5 70.4 80.5 72.5

TGA

GPC

Tg1 (°C)

Tg2 (°C)

W. loss 1 (%)

W. loss 2 (%)

Mw

Mn

Mw/Mn

-37.4

115.3 117.3 100.4 99.8

28 28 20 31

14 26 47 63

43683 39607 68341 188883

27302 23298 36384 72507

1.6 1.7 1.9 2.6

Table 2. Composition and Thermal Properties of the Synthesized Poly(VP-co-AMPS) Copolymers Obtained by 1H NMR, DSC, and TGA composition

poly(VP-co-AMPS) poly(VP-co-AMPS) poly(VP-co-AMPS) poly(VP-co-AMPS)

(30:70) (50:50) (60:40) (70:30)

DSC

TGA

GPC

F(VP)feed

f(VP)copol

w(VP)copol

yield

Tg

W. loss 1 (%)

W. loss 2 (%)

Mw

Mn

Mw/Mn

30 50 60 70

28 47 55 70

17 32 40 56

81.8 83.4 80.1 85.3

135.2 120.9 115.8 113.8

3 21 25 5

43 30 30 44

116882 77857 22102 127840

52021 34652 15238 46597

2.2 2.2 1.5 2.7

is necessary for FGF to recognize their specific tyrosine kinase receptor and to trigger a biological response.6–8 Thus, disruption of the interaction of FGFs with cell surface HSPG seems an evident target for antiangiogenesis. Acidic FGF (aFGF), one of the most abundant FGF isoforms, provides a unique system to explore the molecular basis of new polysulfonated macromolecules as is less stable than basic FGF (bFGF) in the absence of polyanions and requires of a molecule like heparin for significant biological activity. Moreover, aFGF presents low specificity and can complex with a number of sulfonated molecules (e.g., suramin,9–13 suradistas, or pentosan polysulfonate1). These polyanions inhibit the binding of this FGF to the tyrosine kinase membrane receptors and suppress FGFinduced angiogenesis, presenting activity as antitumoral chemotherapeutic agents. The aim of this study was the development of new heparinlike polysulfonated macromolecules based on AMPS to study their effect on the mitogenic activity induced by one of the most relevant pro-angiogenic growth factors, aFGF. AMPS was selected because poly(2-acrylamido-2-methylpropane sulfonic acid) (PAMPS) has been reported as one of the most potent inhibitors of angiogenesis presenting low toxic effects.14 In this study AMPS was copolymerized by radical polymerization with a hydrophilic monomer, VP, or a hydrophobic monomer, BA, to obtain macromolecules with different monomer sequences, and different hydrophilic/ hydrophobic balance. Antimitogenic activity was assayed “in vitro” to determine the monomer distributionbioactivity relationship of the synthesized water-soluble macromolecules.

2. Materials and Methods 2.1. Materials. AMPS (99%), was provided by Avocado and used as received. VP (>99%) and BA (>99%), both from Sigma-Aldrich, were carefully distilled under reduced pressure. Dioxane (Panreac) was refluxed over potassium hydroxide (150 g/L) for 12 h and distilled under nitrogen. Dimethylsulfoxide, DMSO (Scharlau), was used without further purification. Azobis(isobutyronitrile), AIBN (Merck), was recrystallized twice from ethanol. Methacryloylchloride (98%) was distilled under nitrogen before used. 2.2. Synthesis of the Copolymeric Systems. Two different bioactive copolymeric systems based on AMPS were synthesized by free radical copolymerization (Tables 1 and 2). VP or BA was employed as comonomer to obtain polymers with different hydrophilic-hydrophobic balance. Poly(BA-co-AMPS) and poly(VP-co-AMPS) were synthesized in water-dioxane (80:20). For each reaction, the appropriate monomers

Table 3. Reactivity Ratios of the Copolymeric Systems Determined by In Situ 1H NMR reactivity ratios poly(BA-co-AMPS) poly(VP-co-AMPS)

rBA ) 3.68 rVP ) 0.12

rAMPS ) 0.30 rAMPS ) 0.27

were mixed in suitable proportions to obtain a total monomer concentration of 0.5 M. The solution was deoxygenated bubbling pure nitrogen during 30 min. The polymerization reaction was induced thermally at 60 °C using AIBN (1.5 × 10-2 M) as free radical initiator. After 24 h, the polymer was precipitated in acetone and redissolved in water. The solution was then dialyzed (Slide-A-Lyser 3.5K Dialysis Cassette, 3500 molecular weight cutoff, PIERCE) against deionized water for 48 h to minimize the presence of residual unreacted monomers and low molecular weight molecules. Finally, all copolymers were isolated by freeze-drying. 2.3. Polymer Characterization. 2.3.1. NMR Analysis. 1H NMR was performed in an INOVA-400 apparatus operating at 400 MHz. The spectra were recorded using 5% (w/v) DMSO-d6 or D2O (both from Merck) solution, depending on solubility. Copolymer composition was calculated from 1H NMR spectra. 2.3.2. ReactiVity of the Comonomers. The reactivity of the comonomers was evaluated by the estimation of the reactivity ratios (r1 and r2) of the two AMPS-based systems (Table 3), which were determined by in situ 1H NMR, as described by Aguilar et al.15 Briefly, the experiments were carried out in a Varian 400 aparatus. To obtain quantitative data, the following conditions were used: a pulse sequence of 7 µs, equivalent to a 90° tip angle, and a 60 s delay time were applied. The spinning rate of the samples was 7 Hz, and for each datum, only one acquisition (FID), nt ) 1, was registered. The sample temperature was maintained at 60 °C using the heater controller of the NMR equipment. A solution of p-dichlorobenzene (DCB) in DMSO-d6 (10 mg/mL) in a thin wall capillary tube introduced in the NMR tube was used as reference. Signals were integrated using NUTS software (NMR Utility Transform Software) after Fourier transform of the FIDs, and the concentrations were determined as follows:

[AMPS] )

[BA] )

H1 B1 + H1

B1 B1 + H1

(1)

(2)

where H1 corresponds to the integrated peak intensity of the protons assigned to AMPS, and B1 corresponds to the integrated peak

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Garcıá-Fernańdez et al.

Figure 1. Details of the acrylic region of some representative 1H NMR spectra of the poly(BA-co-AMPS) copolymerization reaction at different times with feed molar composition of FBA ) 0.42.

Figure 2. Details of the acrylic region of some representative 1H NMR spectra of the poly(VP-co-AMPS) copolymerization reaction at different times with feed molar composition of FVP ) 0.30.

intensity of the acrylic protons assigned to BA (Figure 1). The values were normalized using the initial feed compositions and the value of the reference peak.

H1 [AMPS] ) V3 + H1

[VP] )

V3 V3 + H1

°C/min, depending on the sample, was used in the analysis. Tg of the polymers was defined as the inflection point of the heat capacity transition. 2.3.4. TGA Experiments. A Perkin-Elmer TGA-7 interfaced to a thermal analysis data system TAC7/DX was used to study the thermal stability of the polymers. Thermograms were obtained under nitrogen atmosphere, between 40 and 550 °C, using a constant heating rate of 10 °C/min. 2.3.5. GPC Experiments. Number and weight average molecular weight and polydispersity of all the specimens were obtained by gel permeation chromatography (GPC), using a Simadzu CTO 20A coupled to a refraction index detector (RID 10A). The system was formed by three PL aquagel-OH 30, 40, and 50 columns (Polymer Laboratories). By flowing a degassed mobile phase, 0.2 M NaNO3, 0.01 M NaH2PO4 buffered solution at pH 9, at 1 mL/min, a GPC diagram was obtained as a function of the elution time. The molecular weight was determined based on the calibration curve obtained from monodisperse polyethylenglycol standard samples with molecular weights between 1000 and 500000 g/mol (Scharlab). Data were analyzed using the Shimadzu LC solution program. 2.4. Analysis of the Bioactivity. The effect of the synthesized polymers on aFGF-driven mitogenesis and quiescent viability of fibroblasts was studied in vitro as previously described by Fernańdez Tornero et al.16 Briefly, Balb/c 3T3 fibroblasts were seeded in 96well plates at a density of 9000 cells/mL (1800 cells/well) using Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% calf serum as culture medium, and incubated during 6 hours. The medium was replaced with DMEM/ Ham’s F-12 (100 µL/well) and incubated at 37 °C and 5% CO2. After 14 h, aFGF (6.40 ng/ mL), 3 KDa heparin (100 µg/mL), and the polymer (in concentrations between 0 and 1 mg/mL) were added to 0.1% BSA in DMEM, 10 µL of the solutions was added to the wells and incubated during 72 h. To fix the cells, 10 µL of glutaraldehyde (25%) was added to all wells and the plate was incubated for 10 min before washing with milli-Q water and drying at 37 °C. Fixed cells were dyed using a crystal violet solution (1 mg/mL) in 50 mM phosphate buffer pH 7.2. The solution was removed after 10 min incubation and cells were washed to remove the dye excess before drying at 37 °C. Finally, 10% acetic acid (100 µL) was added and mixed and differential absorbance (A620 nm-A690 nm) was measured on a Synergi HT Multi-Detection Microplate Reader (Biotek). Cell viability was evaluated in the presence of the synthetic polymers as described above, however, aFGF was not added to the culture medium.

3. Results and Discussion (3)

(4)

where V3 corresponds to the integrated peak intensity of protons assigned to VP, and H1 corresponds to the integrated peak intensity of the acrylic protons assigned to AMPS (Figure 2). The values were normalized using the initial feed compositions and the value of the reference peak. 2.3.3. DSC Experiments. Differential scanning calorimetry was performed with a Perkin-Elmer DSC-7 interfaced to a thermal analysis data system TAC7/DX or a Q-200 TA Instruments. Between 7 and 10 mg of the exhaustively dried samples were placed in aluminum pans and heated from -90 to 160 °C. A constant heating rate of 10 or 20

Two different bioactive copolymeric systems based on AMPS were successfully synthesized. The hydrophilic-hydrophobic balance of the copolymers was controlled by the copolymerization of AMPS with VP (hydrophilic) or BA (hydrophobic). A series of copolymers of each family were prepared by free radical copolymerization with different feed compositions (Table 1 and 2) being all water-soluble polymers. The new materials were purified by precipitation in acetone and dialysis against water. 3.1. Polymer Characterization. 3.1.1. NMR Experiments. H NMR experiments were performed to determine copolymer molar composition (Figure 3). The VP and AMPS mole fractions of the copolymer were calculated by

1

[VP] ) (IV5 /2)/(IA3A4 /6 + IV5 /2)



[AMPS] ) (IA3A4 /6)/(IA3A4 /6 + IV5 /2) The BA and AMPS mole fractions of the copolymer were calculated by

A ) (IA3A4B4B5 - 4 × B)/6

[

[

if

]

r1r2-1/(1-r1)(1-r2)

(6)

[M1]/[M2] ) x, [M10]/[M20] ) x0, [M2] ) y and

B ) (BB6 /3) y ) y0

[BA] ) B/(A + B)

]

[M20][M1] r2/1-r2 [M2] ) [M20] [M10][M2] (r1 - 1)([M1]/[M2]) - r2 + 1 (r1 - 1)([M10]/[M20]) - r2 - 1

() ( x x0

r2/1-r2

629

1 - r2 + (r1 - 1)x 1 - r2 + (r1 - 1)x0

)

[M20] ) y

r1r2-1/(1-r1)(1-r2)

(7)

[AMPS] ) A/(B + A) Integrated peak intensities were employed for these calculations. Tables 1 and 2 list the corresponding mole fractions of the monomer units in the studied copolymers. 3.1.2. ReactiVity of Comonomers. Reactivity ratios are kinetic parameters that give a clear idea of the average composition and the monomer sequence distribution in statistical copolymer systems. They are related directly with the relative reactivity of the corresponding comonomers and, therefore, with the sequence distribution of the comonomers that is incorporated into the growing copolymeric chains. These kinetic parameters (r1 and r2) were determined accurately by in situ quantitative 1H NMR monitoring. This method has been employed in our group successfully for other copolymeric systems,17–19 and the obtained reactivity ratios were in good agreement with those described in the literature and with data for copolymers prepared at low conversion and analyzed by standard methods. Moreover, several advantages were described for this method as the reaction is followed in real-time, instantaneous feed and instantaneous copolymer compositions can be calculated: there is no need to manipulate any sample and a few milligrams of sample are required for the whole study. The method uses a solution of the differential copolymer equation, which defines the terminal model (eq 6) to fit directly the monomer concentration evolution obtained by the in situ quantitative analysis by 1H NMR spectroscopy (Figures 1 and 2).

d[M1] [M1] r1[M1] + M2 ) d[M2] [M2] [M2][M1] + r2[M2]

(5)

being Mi the molar concentration of monomer i, ri the reactivity ratio of monomer i, and Mi0 the initial molar concentration of monomer i. The method considers any given experimental point xt,yt as the starting point for the rest of the reaction. The first 5-10 points were used for each reaction, and the least-squares optimization to eq 7 leads to the optimum r1 - r2 values (Figures 4A and 5A). Poly(BA-co-AMPS) system copolymerized following a moderate ideal copolymerization behaVior (rBArAMPS ) 1; rBA ) 1/rAMPS). This means that the relative rates of incorporation of the two monomers into the copolymers are independent of the identity of the unit at the end of the propagating species. In this system, BA was more reactive than AMPS toward both propagating species (rBA ) 3.6; rAMPS ) 0.28), therefore, the instantaneously formed copolymers initially contained a larger proportion of BA in the copolymer sequences prepared at low conversion or at the beginning of the polymerization reaction. For example, a BA feed molar fraction of 0.2 will give rise to an instantaneous copolymer molar fraction of 0.47 distributed randomly in the macromolecular chains. Figure 4B predicts the evolution of the instantaneous copolymer molar fraction as a function of conversion and feed molar fraction. These 3D graphs were obtained using the algorithm “Conversion” developed in our group in 2004.20 “Conversion” theoretically predicts the course of any binary copolymerization reaction with the conversion from the knowing reactivity ratios and without any approximation (except those considered for the terminal model). From Figure 4B it can be deduced that poly(BA-co-AMPS) system evolves as follows: BA-rich sequences are initially formed and after the consumption of most of BA, chains rich in the less reactive monomer

Figure 3. 1H NMR spectra of the poly(VP-co-AMPS) (50:50) and poly(BA-co-AMPS) (50:50).

630



Figure 4. (A) Diagram of reactivity ratios rBA vs rAMPS obtained, as described in section 3.1.2; solid squares, copolymerization reaction poly(BAco-AMPS) (50:50); empty circles, copolymerization reaction poly(BA-co-AMPS) (80:20). (B) Instantaneous BA copolymer molar fraction for the system poly(BA-co-AMPS) as a function of conversion and BA feed molar fraction. The thick solid lines represent the course of the reactions to obtain the synthesized copolymers ([BA]feed ) 0.25, 0.50, 0.65, 0.75). (C) Monomer distribution scheme of a reaction with an initial composition of [BA]feed ) 0.5; solid circle, BA; empty diamond, AMPS.

Figure 5. Composition diagram obtained with the reactivity ratios determined by in situ 1H NMR for the systems poly(BA-co-AMPS) and poly(VP-co-AMPS).

(i.e., AMPS) are obtained. The scheme of Figure 4C gives an idea of the distribution of monomeric sequences and the composition heterogeneity of the high conversion macromolecules. This modulated the hydrophobic/hydrophilic balance of the copolymers and therefore their solubility and stability in the physiological medium. Reactivity ratios of poly(VP-co-AMPS) system have been previously determined by Kurenkov et al.21–24 However, these kinetic parameters are strongly affected by the nature of the reaction medium. Our copolymers were obtained in water/ dioxane (80:20), whereas Kurenkov’s were synthesized in water, water-ethanol solutions, or DMSO. Therefore, reactivity ratios were calculated to study the copolymerization reaction in these

experimental conditions as reactivity of polar monomers (especially ionic monomers) changes depending on the solvent polarity. Poly(VP-co-AMPS) copolymeric system presented reactivity ratios less than unity for both monomers (rVP ) 0.12; rAMPS ) 0.28), giving rise to an azeotropic copolymerization. Molar fraction in the feed between molar fraction in the copolymer chains (F/f) plot cross the line representing F ) f in the azeotropic point, fAMPS ) 0.55, where the copolymerization occurs without a change in the feed composition (Figure 5). These values demonstrate clearly the effect of donor-acceptor complexation of poly(VP-co-AMPS) in the reaction medium, with an azeotropic point very close to the equimolecular composition of the monomers feed. Feed compositions near the azeotrope yield narrow distributions of copolymer composition except at high conversion where there is a relative drift to VP or AMPS rich sequences depending on whether the initial feed contains more or less than fAMPS ) 0.45. The distribution of copolymer composition becomes wider as the initial feed composition differs more from the azeotropic composition. The system presented a moderate alternating structure (r1r2 ) 0.03), as both kinds of propagating species (macroradicals) preferably added the other monomer. This could be owed to the donor-acceptor character of the reaction and the complexation of both monomers in these experimental conditions, as previously described by Kurenkov et al.24 and earlier commented in this section. Figure 6 represents the evolution of the poly(VP-co-AMPS) system with the conversion. A totally different behavior was observed if compared to poly(BA-co-AMPS) system. At VP feed molar compositions higher than the azeotrope point, the composition of the instantaneously formed copolymeric chains is relatively constant with conversion until most of AMPS is consumed. At the end of the reaction, VP-rich sequences are obtained (Figure 6C). At VP feed molar compositions lower than 0.45 the copolymer composition obtained at low and medium conversions is practically constant until most of VP is



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Figure 6. (A) Diagram of reactivity ratios rVP vs rAMPS obtained as described in section 3.1.2; solid squares, copolymerization reaction poly(VPco-AMPS) (50:50); empty circles, copolymerization reaction poly(VP-co-AMPS) (80:20). (B) Instantaneous VP copolymer molar fraction for the system poly(VP-co-AMPS) as a function of conversion and VP feed molar fraction. The thick solid lines represent the course of the reactions to obtain the synthesized copolymers ([VP]feed ) 0.30, 0.50, 0.60, 0.70). (C) Monomer distribution scheme of a reaction with a initial composition of ([VP]feed ) 0.5; solid square, VP; empty diamond, AMPS.

Figure 7. Experimental Tg data and Fox equation (dashed line); circle, poly(BA-co-AMPS) system; square, poly(VP-co-AMPS) system.

consumed. At high conversions, AMPS-rich macromolecules are obtained. 3.1.3. DSC Analysis. Glass transition temperatures (Tg) of the synthesized homo and copolymers were determined by this technique (Tables 1 and 2). Tg gives an idea of the flexibility of the polymeric chains which influences the interaction with the biological medium. PAMPS presented a Tg of 157.4 °C; poly(butyl acrylate) (PBA) Tg ) -47.1 °C; poly(N-vinylpyrrolidone) (PVP) Tg ) 105.9 °C. Tg of the AMPS-based copolymers, PAMPS, PVP, and PBA, as well as the weight fractions of BA or VP, are shown in Figure 7. The data were treated on the basis of the Fox equation:

W1 W2 1 ) + Tg Tg1 Tg2

(8)

Fox relationship holds well for poly(VP-co-AMPS) system, which means that the contribution of AMPS units to the flexibility of the copolymer chains is additive, and therefore, the specific intramolecular interactions between neighboring VP and AMPS units observed in hydrated systems are established after the hydration process is completed, as previously described by Alencar de Queiroz et al. for dimethylacrylamide (DMAm) and VP copolymeric system.25 Fox equation does not fit the experimental Tg values for poly(BA-co-AMPS) copolymers. Poly(BA-co-AMPS) (75:25) presented two different Tg values (Table 1), which were attributed to the nature of the formed macromolecules. Tg1 (-37 °C) corresponded to the glass transition temperature of the BA-rich macromolecules and Tg2 (100 °C) corresponded to the glass transition of the AMPS-rich macromolecules formed during the course of the polymerization reaction. This is in good agreement with the monomer distribution predicted by the reactivity ratios of this system (Figure 4B). Poly(BA-co-AMPS) (50:50) and poly(BA-co-AMPS) (25:75) thermograms revealed only one Tg close to the PAMPS transition. This is due to the AMPS-rich segments being the Tg of BA-rich molecules not detected under these experimental conditions. 3.1.4. TGA Experiments. Poly(BA-co-AMPS) System. PBA chains decompose in a single weight loss step to give oligomeric hydrocarbon compound at temperatures ranging from 300 to 450 °C with a temperature of a maximum decomposition rate at 410 °C. The TGA curve for PAMPS showed two weight loss stages. The first region (from 150 to 230 °C) is generally interpreted as the release of ammonia due to the imidization reaction between the amide groups of the monomer units. The second region (from 270 to 320 °C) is attributed to the breakdown of the polymer backbone and the imides formed in the first decomposition region. Poly(BA-co-AMPS) copolymers did not present the cyclation stage (140-210°), which indicates that random copolymers were formed during the polymerization process. Thermal degradation of poly(BA-co-AMPS) copolymers took place in two phases: the first one was attributed to

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Figure 8. Thermal degradation of poly(BA-co-AMPS) copolymers; solid line, PBA; dashed line, poly(BA-co-AMPS) (75:25); dot line, poly(BA-co-AMPS) (50:50); short dashed line, poly(BA-co-AMPS) (65: 25); dash-dot line, poly(BA-co-AMPS) (25:75); dash-dot-dot line, PAMPS.

the decomposition of poly(BA-co-AMPS) sequences (from 250 to 360 °C); the second phase (from 350 to 450 °C) was due to the degradation of BA-rich sequences (Figure 8). Actually, weight loss percentage between 350 and 450 °C is closely related to the molar fraction of BA in samples poly(BA-coAMPS) (75:25) and (25:75) and was a bit lower in sample poly(BA-co-AMPS) (50:50; Table 1). Poly(VP-co-AMPS) System. PVP degradation occurred between 350-470 °C with a temperature of maximum decomposition rate of 440 °C. PVP chains decompose by the action of temperature by the random cleavage of the main chains to give rise hydrocarbon compounds of low molecular weight. Thermal stability of these copolymers is higher than PAMPS and between both homopolymers. Heating of poly(VP-coAMPS) copolymers is accompanied by a gradual mass loss from 50 to 150 °C following to a plateau extending to 270-350 °C, depending on the specific sample (Figure 9). This mass loss can be related to the release of water adsorbed by the samples. Further heating leads to a two stage decomposition process that could correspond to the degradation of the macromolecules rich in the alternated sequence of monomers synthesized at low and medium conversions (peak around 350 °C in poly(VP-coAMPS) (50:50) and (60:40)) and the macromolecules rich in VP shown as a shoulder around 425 °C (poly(VP-co-AMPS) (70:30); feed composition > azeotrope)) or macromolecules rich in AMPS (poly(VP-co-AMPS) (30:70); feed composition < azeotrope) around 300 °C. 3.1.5. GPC Experiments. Molecular weight of the analyzed samples was determined by GPC and data are shown in Tables 1 and 2. All copolymers presented molecular weights higher than 30.000 Da that corresponds to the renal filtration threshold. This would facilitate a tumor selective uptake of these polymers when administered intravenously due to the macromolecular nature of the synthesized materials and the hyperpermeability of the tumor vasculature (EPR effect).26–28 However, these polymers were not designed to be injected intravenously, but to be directly injected, as an aqueous solution, in the tumor area or after extraction of the solid tumor due to the well known anticoagulant activity that sulfonated polymers can present in contact with blood.29–31


Figure 9. Thermal degradation of poly(VP-co-AMPS) copolymers; solid line, PVP; dashed line, poly(VP-co-AMPS) (70:30); dash-dot, poly(VP-co-AMPS) (50:50); dash-dot-dot, poly(VP-co-AMPS) (60:40); short dot, poly(VP-co-AMPS) (30:70); dot line, PAMPS.

3.2. Analysis of Bioactivity: Monomer DistributionBioactivity Relationship. FGF and VEGF, the two most meticulously investigated angiogenic factors, are coexpressed in human tumors and are associated with tumor vascularity. Moreover, it has been shown that VEGF expression requires the presence of FGFs,32,33 therefore, FGF itself is potentially a key target for developing antitumoral therapies based on antiangiogenesis. It has been demonstrated that the interaction between FGF and heparin or heparan sulfate is required for bFGF to recognize the specific tyrosine kinase of the cell surface, which is responsible in transducing the presence of FGFs in the cell division signal. In the case of aFGF driven mitogenesis, activation of this growth factor specifically by heparin is a nearabsolute requirement.34 Spivak-Kroizmam et al. stated that heparin cause oligomerization of FGF, thereby indirectly crosslinking and activating the receptor (FGFR) in a strictly liganddependent manner.7 This is why synthetic molecules that are able to interfere with the HSPGs/aFGF/FGFR interaction may act as angiogenic inhibitors. In particular, heparin-mimicking polyanionic compounds as sulfonic acid polymers that are able to compete with HSPGs for aFGF interaction may be expected to hamper the binding of aFGF to the endothelial cell surface with consequent inhibition of its angiogenic capacity.35 In this work, the antimitogenic activity of the synthesized heparin-like macromolecules was assayed as a step to evaluate the bioactivity of these polymers. As it was previously mentioned in the introduction, proliferation assays were performed using acidic fibroblast growth factor (aFGF) as it provides a unique system to explore the molecular basis of new polysulfonated macromolecules. This growth factor is much less stable than bFGF in the absence of polyanions and requires the presence of a molecule like heparin for significant biological activity. Fibroblasts were used in the experiments due to the high sensitivity of these cells to the aFGF activity. Poly(BA-co-AMPS) System. Figure 10 summarizes the mitogenesis assay performed showing the effect of poly(BA-coAMPS) copolymers in the culture medium. All copolymers inhibited mitogenesis as a function of the copolymer concentration and activity was related to the AMPS concentration in the copolymer. In the presence of aFGF, the mitogenic effect


Figure 10. Effect of the poly(BA-co-AMPS) copolymers on Balb/c3t3 fibroblast proliferation; solid symbols, aFGF-induced proliferation activated by heparin; empty symbols, toxicity control, no aFGF was added; triangle, poly(BA-co-AMPS) (25:75); circle, poly(BA-co-AMPS) (50:50); square, poly(BA-co-AMPS) (75:25).

ascribed to the addition of the growth factor, is clearly compensated and at the end inhibited by these copolymers. Poly(BA-co-AMPS) (25:75) presented a sustained inhibition of mitogenesis as a function of copolymer concentration in the culture medium. This copolymer, that incorporated the higher amount of AMPS in its structure, was active at all the assayed concentrations, whereas poly(BA-co-AMPS) (75:25) was not active until a concentration of 125 µg/mL or higher was added to the cells. However, the activity of this copolymer at these concentrations is much higher than that developed by poly(BAco-AMPS) (25:75) and there is an almost total inhibition at a concentration of 1000 µg/mL. Poly(BA-co-AMPS) (50:50) presented an intermediate behavior between poly(BA-co-AMPS) (75:25) and (25:75). Therefore, the antimitogenic activity of the poly(BA-co-AMPS) system was modulated not only by the copolymer concentration in the culture medium, but also by the copolymer composition. Poly(BA-co-AMPS) copolymers had no effect on cells that did not receive aFGF. Additionally, absence of floating cells or other signs of lysis indicated that these polymers are not toxic to these cells (Figure 10). Poly(VP-co-AMPS) System. As it is shown in Figure 11, none of the poly(VP-co-AMPS) copolymers had a clear effect on the fibroblast proliferation (always higher that 80%) in the analyzed concentration interval when aFGF was added to the culture medium. Moreover, absence of floating cells or other signs of lysis indicated that these polymers are not toxic to these cells. Liekens et al.35 described a modeling study that predicted the interaction between bFGF and PAMPS. During polymerization, AMPS develops an asymmetric center at the middle carbon of each acrylamido unit, and a stable intramolecular hydrogen bond could be formed between acrylamido units with equivalent absolute conformation. In this situation, a helical structure with sulfonate groups evenly spaced around 9-10 Å is formed. These authors stated that helical PAMPS could be docked into the heparin-binding site of bFGF, inhibiting both the binding with low affinity HSPGs and high affinity tyrosine kinase FGFRs. For this reason, we considered that the different biological activity developed by the copolymers described in this work was related to the different monomer sequence distribution in the polymeric chains. On the one hand, poly(BA-co-AMPS)


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Figure 11. Effect of the poly(VP-co-AMPS) copolymers on Balb/c3t3 fibroblast proliferation; solid symbols, aFGF-induced proliferation activated by heparin; empty symbols, toxicity control, no aFGF was added; circle, poly(VP-co-AMPS) (30:70); diamond, poly(VP-coAMPS) (60:40); triangle, poly(VP-co-AMPS) (70:30).

copolymeric system presented reactivity ratios that predicted the formation of BA-rich and AMPS-rich molecules during polymerization. Consequently, poly(BA-co-AMPS) copolymers present higher probabilities of presenting long AMPS sequences in their structure, and therefore, higher probabilities to incorporate AMPS units in the appropriate helical conformation to interact with the heparin binding site of the aFGF, hampering the interaction with its receptors. On the other hand, poly(VPco-AMPS) was an azeotropic system (r1 and r2 < 1), and alternating monomer sequences are preferably formed, avoiding the formation of the necessary conformation for the interaction with aFGF. This fact explains and justifies the different effect of poly(VP-co-AMPS) and poly(BA-co-AMPS) systems on the fibroblast mitogenesis when aFGF was added to the medium.

4. Conclusions New AMPS-based copolymers with antiproliferative activity have been synthesized and characterized. The monomeric sequences of the two bioactive copolymeric systems modulates the inhibition of mitogenesis of fibroblasts in the presence of aFGF. Poly(VP-co-AMPS) presented a rather alternating distribution of monomers along the copolymeric chains, whereas poly(BA-co-AMPS) presented a clear tendency for the formation of long block sequences that favor the interaction with FGF and therefore the antiproliferative activity of these polymers. Proliferation of the cultured cells could be modulated not only by varying the copolymer concentration in the medium, but also by varying the copolymer composition and monomer sequence distribution. This work demonstrates the influence of the monomer distribution of the copolymeric systems on the biological activity at the cellular level and shows that poly(BA-co-AMPS) macromolecules are good candidates to be tested as antitumoral “polymer drugs” that are compatible, nontoxic, and soluble in physiological medium. Acknowledgment. Partial financial support from MAT200763355and“RamońyCajalProgramme”isgratefullyacknowledged.

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BM901194E

Antimitogenic Polymer Drugs Based on AMPS: Monomer Distribution

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