Macromolecular HPMA-Based Nanoparticles with ... - ACS Publications

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Macromolecular HPMA-Based Nanoparticles with Cholesterol for Solid-Tumor Targeting: Detailed Study of the Inner Structure of a Highly Efficient Drug Delivery System Sergey K. Filippov,*,† Petr Chytil,† Petr V. Konarev,*,‡ Margarita Dyakonova,§ Christine M. Papadakis,§ Alexander Zhigunov,† Josef Plestil,† Petr Stepanek,† Tomas Etrych,† Karel Ulbrich,† and Dmitri I. Svergun‡ †

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, v. v. i., Heyrovský Sq. 2, 162 06 Prague 6, Czech Republic ‡ European Molecular Biology Laboratory, EMBL c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany § Physik-Department, Technische Universität München, Physik-Department, Fachgebiet Physik weicher Materie, James-Franck-Str. 1, 85747 Garching, Germany S Supporting Information *

ABSTRACT: We report a rigorous investigation into the detailed structure of nanoparticles already shown to be successful drug delivery nanocarriers. The basic structure of the drug conjugates consists of an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer bearing the anticancer drug doxorubicin (Dox) bound via a pH-sensitive hydrazone bond and a defined amount of cholesterol moieties that vary in hydrophobicity. The results show that size, anisotropy, and aggregation number Naggr of the nanoparticles grows with increasing cholesterol content. From ab initio calculations, we conclude that the most probable structure of HPMA copolymer−cholesterol nanoparticles is a pearl necklace structure, where ellipsoidal pearls mainly composed of cholesterol are covered by a HPMA shell; pearls are connected by bridges composed of hydrophilic HPMA copolymer chains. Using a combination of techniques, we unambiguously show that the Dox moieties are not impregnated inside a cholesterol core but are instead uniformly distributed across the whole nanoparticle, including the hydrophilic HPMA shell surface.

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INTRODUCTION

tumors due to the enhanced permeation and retention (EPR) effect.3 Passive accumulation of HPMA-based polymer carriers in solid tumors strongly depends on their molecular weight or, more precisely, their coil size in aqueous solutions.4−8 The size of the carriers can easily be increased using supramolecular structures formed by self-assembly of amphiphilic copolymers with molecular weights lower than the limit of renal filtration. The hydrophobic core of the micelle is enveloped with a hydrophilic layer composed of a hydrophilic polymer, which protects the whole system from aggregation and undesired interactions with the components of living organisms. Recently, the synthesis and physicochemical and preliminary biological properties of new self-assembled drug delivery systems based on linear HPMA copolymers containing hydrophobic substituents randomly distributed along the

Polymer drug carriers based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been studied extensively over the last 30 years.1 Most HPMA copolymer− drug conjugates were developed for the treatment of tumors, with a special focus on site-specific delivery and controlled release of anticancer agents into tumor tissues or cells. Proper selection of the polymer carrier size and structure is crucial for obtaining an effective tumor- or tumor cell-specific targeting molecule and enabling intracellular controlled drug release, which is important for the subsequent antitumor activity of the drug.1,2 The antitumor activity of HPMA-based drug conjugates can be improved by enhancing their “passive” tumor accumulation using high-molecular-weight (HMW) carriers or nanosized particles. The high molecular weight or size of polymer carriers prevents fast elimination of the drug from the organism by renal filtration, enabling prolonged blood circulation and retention of the drug in the body. Furthermore, macromolecules are more effectively accumulated in solid © 2012 American Chemical Society

Received: June 2, 2012 Revised: July 5, 2012 Published: July 16, 2012 2594

dx.doi.org/10.1021/bm3008555 | Biomacromolecules 2012, 13, 2594−2604

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polymer chain were described.9,10 The basic structure of the drug conjugates consists of an HPMA copolymer bearing the anticancer drug doxorubicin (Dox) bound via a pH-sensitive hydrazone bond (designed for controlled drug activation in tumor cells or tissue) and a defined number of moieties of varying hydrophobicity (e.g., cholesterol, oleate, dodecyl). It was demonstrated that the presence of a cholesterol moiety results in much slower blood clearance, significantly higher tumor accumulation, and enhanced antitumor activity of the polymer drug when compared with free drug or other polymer drug (Dox) conjugates of linear or grafted structure with increased molecular weight. Moreover, it was described that the treatment of selected tumors (mouse EL-4 lymphoma) with low dose leads up to 100% long-term survival.10 The main advantage of the above-described drug delivery system consists in an easy preparation of polymer precursors, the simple formation of a self-assembled system based on direct dissolution of the polymer precursor in the final PBS buffer and the significantly enhanced antitumor efficacy due to the EPR effect. Nevertheless, a number of fundamental questions remain unanswered: Under what conditions are nanoparticles formed in aqueous solution? Which parameters does the nanoparticle shape depend on? Do the nanoparticles have an internal structure? How does the introduction of Dox alter the properties of the nanoparticles? The vast majority of recent publications devoted to drug delivery by polymeric nanoparticles focus on two issues: (i) the creation of an appropriate polymer for drug delivery that will self-assemble into a nanocontainer and (ii) biological tests of these carriers on laboratory animals. The detailed mechanism of drug delivery and the interior structure of the polymeric nanoparticles have either been out of the scope of researchers or have only been postulated, even though their knowledge is of primary importance for designing efficient drug delivery vectors. Investigations of prospective nanoparticles in aqueous solution using more sophisticated methods are thus needed. Surprisingly, there is a lack of such studies for clinically relevant systems except for recent works by Paul et al.11,12 Here, we report, for the first time, a rigorous, detailed structural investigation of nanoparticles already classified as successful drug delivery nanocarriers. We have completely revised previous results10 and have performed new experiments at a more sophisticated level. Fluorescence correlation spectroscopy (FCS) has allowed us to determine the polymer concentration range in which nanoparticles are formed and reveals their hydrodynamic radius. An advanced dynamic light scattering apparatus was exploited together with additional complementary experimental methods small-angle X-ray and neutron scattering (SAXS and SANS) that probe smaller distances (higher q) in concert with light-scattering methods. We reconstructed the three-dimensional structure of nanoparticles that occur in solutions of HPMA copolymer− cholesterol conjugates. The amount of cholesterol was varied between 0 and 3.0 mol %; conjugates with higher cholesterol content were not soluble in water. Information on the size, shape, and internal structure is presented for conjugates with varying cholesterol content. On the basis of the obtained data, we attempt to explain the advanced antitumor activity of HPMA copolymer−Dox conjugates.

Article

MATERIALS AND METHODS

Synthesis of Monomers. N-(2-Hydroxypropyl)methacrylamide (HPMA), 6-methacrylamido hexanoyl hydrazine, and cholest-5en-3βyl 6-methacrylamido hexanoate were synthesized as described.10,13,14 N-(2-Hydroxypropyl-1,1,2,3,3,3-d6)methacrylamide (HPMA-d6) was synthesized by similar reaction procedure as HPMA: 392 mg (3.7 mmol) of anhydrous sodium carbonate was suspended in a solution of 300 mg (3.7 mmol) of (±)-1-amino-2-propanol-1,1,2,3,3,3d6 in 1 mL of freshly distilled methylene chloride. A solution of 386 mg (3.7 mmol) methacryloyl chloride and an inhibitor octylpyrocatechine in 1 mL of methylene chloride was added dropwise under vigorous stirring within 0.5 h. After 1 h, 570 mg of anhydrous sodium sulfate was added and the solid was filtered off. HPMA-d6 was obtained by crystallization from methylene chloride at −20 °C and purified by recrystallization from acetone. Yield 72%; mp 65−66 °C; 1H NMR [acetone-d6]: δ 7.26 (br, 1H (OH)), 5.74 and 5.34 (d, 2H (CH2)), 4.08 and 2.93 (br, 1H (NH)), 1.94 (s, 3H (CH3)). 13C NMR [acetone-d6]: δ 18.13 (CH3), 19.32−20.07 (CD3), 45.96−47.08 (CD2), 64.16−74.37 (CD), 118.46 (CH2C), 140.69 (CCH2), 168.39 (CO). The structure and purity of the monomers were examined by 1H NMR (Bruker spectrometer, 300 MHz) and by HPLC (Shimadzu 10VP) using a C18 reverse-phase Chromolith Performance RP-18e (4.6 × 100 mm) column with diode array detection. The eluent was water−acetonitrile with a gradient of 5−95 vol% acetonitrile, 0.1% TFA, and a flow-rate of 1 mL·min−1. Synthesis of Polymer Precursors. Polymer precursor I was synthesized by solution radical copolymerization of HPMA and 6methacrylamido hexanoyl hydrazine in methanol using AIBN (Azobisisobutyronitrile) as initiator: AIBN (1 wt %), monomers (14 wt %), molar ratio HPMA/6-methacrylamido hexanoyl hydrazine 93:7. Reaction conditions and isolation of polymers were carried out as described.14 Polymer precursors II, IV, V, VI, and VII were prepared by solution radical terpolymerization of HPMA, 6-methacrylamido hexanoyl hydrazine and cholest-5en-3β-yl 6-methacrylamido hexanoate in methanol using AIBN as initiator: AIBN (1 wt %), monomers (14 wt %), molar ratio HPMA/6-methacrylamido hexanoyl hydrazine/ cholest-5en-3β-yl 6-methacrylamido hexanoate 90.5:8:1.5 (II), 89.6:8:2.4 (IV), 89:8:3 (V, VI), 88.8:8:3.2 (VII). Reaction conditions and isolation of polymers were carried out as described.10 Polymer precursor III was prepared similar to polymer IV using HPMA-d6 instead of HPMA. Synthesis of Polymer−Dox Conjugates. Polymer−Dox conjugates I-Dox, II-Dox, IV-Dox, and VI-Dox were prepared by the reaction of corresponding polymer precursors I, II, IV, and VI containing hydrazide groups with Dox in methanol in the dark.15 The polymer−drug conjugates were purified from low molecular weight impurities (Dox or its degradation products) by gel filtration using a Sephadex LH-20 column and methanol as an eluent. FCS. All FCS measurements were performed using a ConfoCor2 from Carl Zeiss Jena GmbH. It is equipped with an Ar+ laser operated at 488 nm, a pinhole of diameter 80 μm, a C-Apochromat 40x/1.2 water immersion objective, a BP 530−600 bandpass emission filter, and an HFT 488 plate dichroic beam splitter. A Lab-tek 8-well chambered coverglass from Nalge Nunc International was used as a sample chamber. The measurement time was 60−120 s. The measurements were repeated 15−30 times, and the correlation functions are averaged. This procedure was repeated 2−3 times to confirm the results. The laser power was attenuated to 0.1−1.0% of the maximum power of 200 mW to avoid bleaching of the dye molecules. The count rate was approximately 30−100 kcts/s, depending on the attenuation. The resulting autocorrelation curves were fitted by the following equation:16 2595


Biomacromolecules G(τ ) = 1 +

⎡ ⎛ τ ⎞⎤ TT 1 × ⎢1 + exp⎜ − ⎟⎥ × N ⎢⎣ 1 − TT ⎝ τT ⎠⎥⎦

n

scattering by normalization against reference solutions of bovine serum albumin. DAMMIF,22 a fast version of DAMMIN,23 was used to reconstruct the low-resolution shape of nanoparticles. The results of 20 DAMMIF runs were averaged to determine common structural features using DAMAVER24 and SUPCOMB.25 Prior to the DAMMIN/DAMMIF calculation, an appropriate constant was subtracted from each data point to force the q−4 decay of the intensity at higher angles following Porod’s law26 for homogeneous particles with a smooth surface. This procedure yields a “shape scattering” curve corrected for the unwanted scattering contribution from the internal structure. To check the robustness of the PDDF calculations, the GIFT (generalized indirect Fourier transformation) software provided by O. Glatter27 was used as an alternative. Dynamic and Static Light Scattering (DLS and SLS). Measurements were carried out on an ALV instrument equipped with a 22 mW He−Ne laser in the angular range 30−140°. Measurement times were selected for each concentration to accumulate a smooth correlation function. The SLS data were analyzed using the Zimm plot procedure. The obtained correlation functions were analyzed by REPES,28 an analytical software providing the distribution function of hydrodynamic radii, G(Rh). To account for the logarithmic scale on the Rh axis, all DLS distribution diagrams are shown in the equal area representation, RhG(Rh).29 In all experiments, about 2 mL of the sample was filtered by 0.22 μm poly(vinylidene difluoride) filters and transferred to a sealed dustfree light-scattering cell. The temperature was controlled within 0.05 °C. As all the observed modes were diffusional in nature (1/τ = Dtq2), the values of the translation diffusion coefficients Dt were calculated from the slope of the relaxation rate 1/τ versus q2 (Supporting Information, Figure S1). The apparent hydrodynamic radius of the nanoparticles, Rh, was calculated using the Stokes−Einstein equation. The refractive index increments, dn/dc of the substances were measured with a Brice-Phoenix differential refractometer at a light wavelength of 633 nm. SANS. SANS experiments were performed at CEA-Saclay on the PAXY of the Laboratoire Leon-Brillouin. Measurements were run on a 128 × 128 multidetector (pixel size 0.5 × 0.5 cm) using a nonpolarized, monochromatic (wavelength λ set by a velocity selector) incident neutron beam collimated with circular apertures for two sample-to-detector distances, namely, 1 m (with λ = 0.6 nm) and 7 m (with λ = 0.8 nm). With this setup, the investigated q-range was 4 × 10−2 to 4 nm−1. In all cases, the two-dimensional scattering patterns were isotropic and were azimuthally averaged resulting in the dependence of the scattered intensity Is(q) on the scattering vector q. The curves were corrected for background scattering and detector efficiency. The intensities of neutron scattering are given in arbitrary units.

ρi

∑ i=1

Article

(1 + )(1 + τ τD, i

τ 1 (z 0 / w0)2 τD, i

1/2

)

(1)

where N is the total number of fluorescent particles in the observation volume, n is the number of different fluorescent species, τD,i is the diffusion time of the ith species, ρi is the amplitude of the ith species, and z0 and w0 are the half-height and half-width of the observation volume, respectively. TT and τT are the triplet fraction and time of the fluorescent dye, respectively. From the fit of 1 or 2 decays (n = 1 or 2), the triplet values were found to be in the range TT = 0.1−0.2 and τT = 1−3 μs. w0 was determined before each session by measuring the diffusion time of rhodamine 6G (Sigma-Aldrich, DRh6G = 2.8 × 10−10 m2 s−1),17 τD,Rh6G, and by using the relation w0 = (4DRh6G τD,Rh6G)1/2. A value w0 ≅ 0.2 μm was obtained. The ratio z0/w0 determined from the fit to the Rh6G correlation function in water was typically 5−6. The hydrodynamic radius of the diffusing particles was determined using the Stokes− Einstein law. For the FCS measurements, polymer VII was dissolved in phosphate buffer saline (PBS) (pH 7.2) at a concentration of 2.8 × 10−4 g·mL−1. This solution was stepwise diluted with PBS buffer to obtain polymer concentrations down to 2.8 × 10−6 g·mL−1. All solutions were stirred for 2 days at room temperature. Then, an aqueous solution of the fluorescent dye (Rhodamine 6G) was added to all solutions such that its concentration in the final solution was 0.479 × 10−8 g·mL−1. A second series of solutions was prepared by subsequently adding small amounts (20−50 μL) of PBS buffer to a VII/Rh6G solution prepared as described above, which had a concentration of 8.4 × 10−5 g·mL−1 directly in the FCS cell. All measurements were carried out at room temperature (18−22 °C). SAXS. A series of SAXS experiments were performed at the beamline X33 at the EMBL, HASYLAB (Hamburg, Germany).18 The SAXS setup is based on a pinhole camera with a beam stop placed in front of a two-dimensional detector (1 M PILATUS). The X-ray scattering patterns were recorded for sample-to-detector distance 2.7 m, using a monochromatic incident X-ray beam (λ = 0.15 nm) covering the range of momentum transfers 0.06 < q < 6.0 nm−1 (q = 4π sin θ/λ, where 2θ is the scattering angle). No measurable radiation damage was detected by comparison of eight successive time frames with 15 s exposures. In all the cases reported in this paper, the twodimensional scattering patterns were isotropic. They were azimuthally averaged to yield the dependence of the scattered intensity Is(q) on the scattering vector q. The scattering intensities were put on absolute scale using BSA solution. Prior to fitting analysis, the solvent scattering had been subtracted. Another set of SAXS experiments was performed using a pinhole camera (Molecular Metrology SAXS System) attached to a microfocused X-ray beam generator (Osmic MicroMax 002) operating at 45 kV and 0.66 mA (30 W). The camera was equipped with a multiwire, gas-filled area detector with an active area diameter of 20 cm (Gabriel design). Two experimental setups were used to cover the q range of 0.07−11 nm−1. The scattering intensities were put on absolute scale using a glassy carbon standard. Prior to fitting analysis, the solvent scattering had been subtracted. The background from air and the capillary was also subtracted. All data manipulations were performed by using the PRIMUS software.19 Data were extrapolated to zero concentration of solute to get rid of interparticle interaction influence. The forward scattering I(q = 0) and the radius of gyration Rg were evaluated using the Guinier approximation.20 These parameters were also computed from the entire scattering patterns using the computer program GNOM,21 which provides the pair distance distribution functions [PDDF(r)] from which the shape of the nanoparticles was estimated and the maximum particle dimension (Dmax) was determined. The molecular weights (Mw,exp) of the solutes were estimated from the forward

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RESULTS

We studied drug conjugates consisting of N-(2-hydroxypropyl)methacrylamide copolymer bearing the anticancer drug doxorubicin (Dox) bound via a pH-sensitive hydrazone bond as well as a defined amount, varying from 0 and 3.0 mol %, of cholesterol moieties differing in their overall hydrophobicity. For selected polymers, Dox moieties were attached to a polymeric backbone. Thus, we can monitor continuous changes in morphology and physicochemical properties of nanoparticles with growing cholesterol content formed at two different pH values and assess the effect of the inclusion of Dox into the polymeric chain. Synthesis of Polymer Precursors and Their Drug Conjugates. Polymer precursors differing in cholesterol content were synthesized by terpolymerization of HPMA with comonomers bearing hydrazide groups and cholesterol moieties. Copolymer III was synthesized in a similar manner to IV using partially deuterated HPMA-d6. No difference in 2596


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concentrations above the CMC, the dye is attached to micelles, and the micellar diffusion coefficient is measured.31,32 We focus here on polymer VII, which contains 3.0 mol % of cholesterol. For concentrations below 1.68 × 10−5 g·mL−1, a single decay (eq 1) is fitted to the normalized fluorescence autocorrelation functions, shown in Figure 2. In contrast, two

physicochemical properties between III and IV was observed. As a reference, copolymer I bearing no cholesterol molecules was synthesized. The polymerization yields varied between 65 and 75%. The composition of the terpolymers (∼6 mol % of hydrazides; for cholesterol content, see Table 1) approximately Table 1. Physico-Chemical Characteristics of the Polymers sample

z (content of cholesterol groups), mol %

content of Dox, wt %

Mw, g/mol

Mw/Mn

I I-Dox II II-Dox III IV IV-Dox V VI VI-Dox VII

0 0 1.4 1.4 2.3 2.3 2.3 2.7 2.8 2.8 3.0

0 10.0 0 8.6 0 0 10.1 0 0 8.7 0

18770 25000 16300 23700 18500 17200 21100 16700 16900 24400 28000

1.83 1.90 2.20 1.97 1.76 1.92 1.75 1.99 2.15 2.17 1.73

Figure 2. Normalized FCS correlation functions of conjugate VII at pH 7.2 at room temperature. Conjugate concentrations are given in the graph.

corresponded to the composition of the polymerization mixture; the copolymerization parameters did not differ substantially from unity. The polymer precursors I−VI were used for conjugation with Dox. The reaction resulting in the hydrazone bond-linked Dox was not influenced by the presence of cholesterol introduced into the polymer structure. The polymer precursors were of similar molecular weight as measured by SEC in an 80:20 mixture of methanol/acetic buffer, which was assumed to disrupt any hydrophobic interactions. Critical Micelle Concentration (FCS). The low concentration regime was addressed using FCS, which allows the critical micelle concentration (CMC) of amphiphilic polymers to be determined.30 Adding a fluorescent dye to a polymer solution of varying low concentration reveals the presence of hydrophobic compartments: At polymer concentrations below the CMC where only polymeric monomers are present, only the freely diffusing dye is detected. In contrast, at polymer

decays are needed above that concentration. At concentrations above 2.8 × 10−4 g·mL−1, particles of a few 100 nm in hydrodynamic radius are observed, indicating the formation of large aggregates. Because their size cannot be determined reliably with the present setup, these high concentrations are not considered further. Figure 3 shows the resulting hydrodynamic radii as well as the relative amplitude of slow decay. Up to 1.7 × 10−5 g·mL−1, a single diffusional decay is observed with Rh ≅ 1 nm (Figure 3a). We attribute this process to the freely diffusing dye Rh6G. Above this concentration, an additional slower process is observed with increasing amplitude (Figure 3b). The CMC was thus determined to be 2.1 × 10−5 g·mL−1. The corresponding hydrodynamic radii increase with polymer concentration from 11.2 to 21.4 nm. Notably, a plateau is observed at ∼4 × 10−5

Figure 1. Schematic structures of the polymer precursors (a) and polymer−drug conjugates (b) bearing cholesterol moieties. For conjugate III (see Table 1), deuterated hydrogens are bonded to carbon atoms marked with *. 2597


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Figure 4. Scattered intensity Is extrapolated to infinite dilution as a function of the scattering vector q for polymers I−VII. Inset: Scattered intensity Is as a function of the scattering vector q for polymer VI at pH = 5.0; Black curve, c = 2 × 10−2 g·mL−1; green curve, c = 1 × 10−2 g·mL−1; blue curve, c = 5 × 10−3 g·mL−1, red curve, c = 2 × 10−3 g·mL−1.

nearly the same for all polymers regardless of cholesterol content. The scattering curves were almost identical at q > 1.5 nm−1. In contrast, the intensity at low q increases with increasing cholesterol content (Figure 4), which can be regarded as indirect evidence of nanoparticle growth assuming that the changes of contrast are negligible. Model-independent assessment should be performed prior to any model selection for the fitting of scattering profiles. The SAXS technique is commonly used to extract modelindependent information regarding the conformation and size of macromolecules in solution that can be performed by the analysis of Kratky or Guinier plots.33 Scattering from macromolecules in solution usually reveals three regions in the dependence of scattered intensity on the scattering vector I ∼ qα, with characteristic behaviors for various length scales of the chain. These are the Guinier regime (qRg < 1), the regime characterizing the coil conformation (self-avoiding random walk conformation) with α = −2 in θ-solvents and α ≈ −5/3 in good solvents, and the region at higher q behaving as ∼q−1, revealing the local stiffness of the macromolecule. In contrast, for compact nanoparticles, Porod behavior ∼q−4 is typically observed in the intermediate and high q-range. Low q Range (Guinier Analysis). The Rg value was extracted by applying the Guinier approximation I(q) = I(0) exp(−Rg2q2/3) to the initial region of SAXS curves for all polymers at four concentrations. The advantage of this approach is that it can be used regardless of the shape of the object involved. An extrapolation of the Rg values to infinite dilution was subsequently performed, as shown in Figure 5, for selected conjugates. All dependences could be approximated as linear. Two features should be noted: (i) that the intercept of the curves increases with the cholesterol content of the macromolecules, indicating that they grow and (ii) that whereas all Dox−free conjugates have a negative slope, the presence of Dox in a polymer structure changes the slope to a positive value (Figure 5). The variation of the apparent radius of gyration Rg with concentration is the standard manifestation of intermolecular interactions described by a second virial coefficient A2 in the approximation of pairwise interactions. In the Zimm

Figure 3. Results from FCS on conjugate VII at pH 7.2 at room temperature: (a) hydrodynamic radii and (b) fraction of the slow decay as a function of conjugate concentration in PBS buffer. Black circles are obtained as described in the Materials and Methods, and red triangles are obtained by diluting a solution of 2.8 × 10−5 g·mL−1 with PBS buffer directly in the FCS sample cell. The vertical dashed line indicates the CMC.

g·mL−1, indicating a nonmonotonous growth process of the nanoparticles. Above the CMC, the fraction of the slow decay increases from 0.2 to 0.6 with increasing polymer concentration, indicating an increase of the fraction of the micelles in the solution. We thus conclude from FCS that nanoparticles are formed above the CMC. Nanoparticle formation is presumably driven by the hydrophobic cholesterol moieties. The plateau of the hydrodynamic radius indicates that existing nanoparticles associate rather than to grow continuously. Nevertheless, the detailed structure of the nanoparticle remains ambiguous. Shape and Solution Properties (SAXS). To investigate the potential spatial structure of nanoparticles in solution, we performed SAXS experiments. SAXS curves for polymers without Dox at concentrations of 2 × 10−3, 5 × 10−3, 1 × 10−2, and 2 × 10−2 g·mL−1 were collected (inset of Figure 4). The solutions of conjugates that bear Dox were measured at concentrations 1 × 10−3, 2.5 × 10−3, 5 × 10−3, and 1 × 10−2 g·mL−1. As mentioned above, we extrapolated the scattering curves to infinite dilution to suppress the influence of the structure factor S(q). Figure 4 shows the extrapolated scattering curves for some conjugates. Two local minima and maxima are visible in the scattering curves for conjugates that bear cholesterol moieties. Polymer I without cholesterol had only one maximum and minimum at high-q range. Notably, the extreme points are 2598


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hypothesis was confirmed by static light scattering experiments (see below). Intermediate q Range (Kratky Plot). The inner structure of the nanoparticles (compact or branched polymer structures) is easily detectable in a Kratky plot (I(q) q2 vs q). For hard spheres, a pronounced maximum is followed by multiple peaks,33 whereas a plateau is observed for branched systems: I(q)q2 for a polymer in Gaussian conformation steadily grows, reaching a plateau without any peak.33 The peak position is expected to be inversely proportional to the radius of gyration. The scattering curves extrapolated to infinite dilution in the Kratky representation are provided in Figure 7 for some of the

Figure 5. Plots of Rg as a function of concentration c for conjugates: (Δ) I, pH = 7.2; (○) II, pH = 5.0; (□) VII, pH = 5.0; (●) II-Dox, pH = 5.0; (▲) IV-Dox, pH = 5.0; (■) VI-Dox, pH = 5.0.

approximation,34,35 the slope of Rg(c) is given by −2A2Mw. A negative slope for all Dox−free conjugates indicates that the interactions between polymer segments and solvent molecules are energetically favorable; the A2 value is positive. The presence of hydrophobic Dox in the polymer chain unambiguously changes the solvent quality to make it a poor solvent; A2 is negative. We can expect that a polymer chain with Dox has a tendency to contract in solution. Sample I (polymer precursor), which bears neither cholesterol nor Dox, has a slope which is nearly zero (Figure 5). The polymer most likely assumes an ideal Gaussian conformation at the θ condition. The dependence of Rg extrapolated to infinite dilution as a function of cholesterol content is presented in Figure 6 for

Figure 7. Kratky plot for I, I-Dox, II, IV, IV-Dox, and VII (pH = 5.0). The scattering curves were extrapolated to infinite dilution.

conjugates; changes of I(q)q2 with increasing cholesterol content are clearly discernible in the intermediate q range. The polymer precursor I without cholesterol moieties has the features expected for a typical linear macromolecule in a Gaussian conformation: I(q)q2 plateaus with no oscillations (Figure 7). Such behavior corresponds to a q−2 law that is also detectable in the log−log plot (Figure 8). Calculation of the slope of the Is(q) versus q dependence in the intermediate q range gives −2.00 ± 0.05 and −2.19 ± 0.02 for the conjugates I and I-Dox, respectively. It was previously shown33 that such a dependence (q−2) is observed only if the conformation of a polymer is truly Gaussian (in a θ-solvent); it becomes lower or higher if an excluded volume effect exists

Figure 6. Dependence of Rg0 as a function of cholesterol content for Dox-free (●, pH = 5.0; □, pH = 7.2) and Dox-modified conjugates (red circle, pH = 7.2).

Dox-free conjugates. The dependence follows a linear law. Nanoparticles composed of conjugates with higher amounts of cholesterol have larger sizes. A similar tendency is observed for conjugates with Dox (Figure 6). Nanostructures composed of Dox-modified conjugates are generally slightly smaller. We suggest that the hydrophobic Dox affects the intrapolymer interactions such that the nanoparticles appear more compact because of the contraction of a single polymer chain. This

Figure 8. Log−log plot for the conjugates I and I-Dox (pH = 7.2). The scattering curves were extrapolated to infinite dilution. 2599


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In contrast, the PDDF of a cylinder displays a straight asymmetrical tail.33 The effect of the cholesterol content on the size of nanoparticles can best be observed in real space in the PDDF shown in Figure 9. All curves show a pronounced peak at low r-

between segments such that the distribution of segments is non-Gaussian.33 We conclude that conjugate I has a truly Gaussian conformation, as suggested earlier. In contrast, conjugate I-Dox, which bears 10 wt % of Dox, has an excluded volume effect on polymer conformation. The −2.19 slope value indicates that attractive forces between polymer segments dominate. The nonideality of the Gaussian conformation is also detectable in a Kratky plot where a slight decrease of I(q)q2 is observed at q > 1.3 nm−1 (Figure 7). This is not surprising if we recall the hydrophobicity of Dox. Obviously, hydrophobic Dox perturbs the ideal Gaussian conformation, resulting in a more compact polymeric coil. The predominance of attractive pair interactions between polymer segments over polymer−solvent ones is in agreement with the negative value of the second virial coefficient reported above. A further increase in the content of Dox moieties is expected to cause contraction of the macromolecule and precipitation. Nevertheless, we observe that, even at 10 wt % of Dox, the I-Dox conjugate still has a coil conformation with an excluded volume effect where attractive forces between polymer segments dominate. The Kratky plot clearly demonstrates that the introduction of cholesterol, which is much more hydrophobic than Dox, into a polymer chain results in the formation of compact objects (Figure 7). The corresponding peak is present in the Kratky plot for the conjugates II, IV, and VII with varying cholesterol content. We thus have a more direct proof of nanoparticle formation and that no coil conformation remains. The change in pH from 7.2 to 5.0 does not alter the nanoparticle structure; the peak is still manifested in the Kratky plot. The inclusion of Dox moieties does not hinder nanoparticle formation (green line, Figure 7). However, the Kratky representations unequivocally reveal structural differences between conjugates with and without Dox at the same amount of cholesterol. Compared to conjugate IV, the peak for the conjugate IV-Dox is shifted to higher q values, which is indicative of a smaller size in comparison with the Dox-free conjugate IV (blue line in Figure 7). This conclusion is in agreement with data from the Guinier analysis reported earlier (Figure 6). However, the minimum that follows the peak is less pronounced for the IV-Dox conjugate. Because the minimum depth is related to the surface sharpness, we conclude that the Dox containing conjugates have less sharp boundaries than the Dox-free conjugates. GNOM/GIFT Analysis. To gain a deeper understanding of nanoparticle structure, we exploited the GNOM/GIFT software to calculate the pair distance distribution function (PDDF) function. The PDDF is given by the inverse Fourier transformation of the form factor P(q) of a nanoparticle: PDDF(r ) = 4π

∫0

∞

P(q)

sin qr dq qr

Figure 9. Pair-distance-distribution function for certain conjugates. The scattering curves were extrapolated to infinite dilution. Arrows indicate shoulders on the PDDF curves.

values that is followed by asymmetric tails for higher r-values. Such curves indicate an elongated nanoparticle shape. The PDDF curves clearly show that the maximum dimension of nanoparticles, Dmax, increases with increasing cholesterol content in the conjugates, which is in agreement with the Rg data reported earlier. More interestingly, a higher cholesterol content results in a more asymmetric tail of the PDDF function. From Figure 9, we conclude that the conformation of the nanoparticles evolves. Thus, conjugates with low cholesterol content (II and IV) have a shape that is close to a prolate ellipsoid. In contrast, for the conjugate with 3.0 mol % (VII), the PDDF curve indicates a nearly rod-like structure. Meanwhile, some shoulders are present on the PDDF curves for VI and VII conjugates (Figure 9). Given that the maxima in all PDDF functions are at the same position (at about 6 nm, Figure 9), it is clear that the evolution of the structure proceeds via its elongation, which makes a pearl-necklace model the most plausible model for such polymers. Again, the pH only has a minor influence on the shape of nanoparticles (Figure 9). The introduction of Dox changed the PDDF curves somewhat. While the maximum dimensions Dmax for Dox-free and Dox-modified conjugates are nearly the same (Figure 10), closer inspection reveals evidence for some structural changes with the inclusion of Dox. The peak shift indicates a lower radius of gyration, whereas the asymmetric tail at large distances is smoother for the conjugates IV and IV-Dox than for II and IIDox (Figure 10). A more uniform behavior of the PDDF function at large distances suggests that the thickness of the elongated particle formed by conjugate IV-Dox is more homogeneous, making it a truly rod-like particle. At this stage, we speculate that the Dox moieties perturb the pearls by preserving the end-to-end distance but lowering the Rg value. Having obtained the proof of cylindrical symmetry for the nanoparticles, we can calculate the so-called cross-sectional pair-distance distribution function, pc(r) (Figure 11). The cross-section PDDF is a standard tool utilized to gain information about the radius of gyration of a particle’s crosssection, Rg,cs, and has been extremely useful for bottle-brush

(2)

The PDDF provides model-independent information for particle structures in real space.33 The PDDF curve obtained from such analysis is conformation-sensitive with theoretically known shapes for different models.34 For spherical conformations, a classical bell shape should be obtained by the inverse Fourier transformation. For an elongated particle, an asymmetrical tail is discernible at large distances, r. If several prolate ellipsoids are merged together, a PDDF for the final structure will manifest several bumps, with a higher number of constituent elllipsoids resulting in a higher number of bumps. 2600


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nanoparticles have heterogeneous thicknesses that correlate with the pearl-necklace structure proposed above. DAMMIN/DAMMIF Calculations. We utilized the DAMMIN software,36,37 an ab initio method, for the reconstruction of the particle shape and the internal structure from small-angle scattering data. This model begins with a particle built from a sphere of densely packed dummy scattering centers. By means of subsequent annealing38 of those scattering centers, a configuration is found that fits the scattering data. An advantage of this approach is that such calculations require no a priori knowledge of the particle architecture. The single parameter required for simulation is the maximum particle size. It has been shown23 that this method correctly reconstructs the general shape of a number of biological objects. The results of simulations on our nanoparticles are shown in Figure 12. The Figure 10. Pair-distance-distribution function for the polymers II, IIDox, IV, and IV-Dox at pH = 5.0 and 7.2. The scattering curves were extrapolated to infinite dilution.

Figure 11. Cross-sectional pair-distance-distribution functions pc(r), for the Dox-free conjugates II, V, VI, and VII at pH = 7.2. The scattering curves were extrapolated to infinite dilution. Figure 12. Hypothetical nanoparticle structures modeled by DAMMIN/DAMMIF for conjugates II, V, and VII. For these calculations, the scattering curves were extrapolated to infinite dilution.

polymers.33 An analysis (Figure 11) proved that the Rg,cs values of the conjugates increases with z, the amount of cholesterol groups (see Table 2). However, none of the curves has a constant cross-section over a large r range; rather several distinctive bumps are present, which we attribute to a variation of the cross-section of Rg. This finding indicates that all

stability of the calculations was inspected by performing 20 runs, and the DAMMIN/DAMMIF calculations proved our hypothesis. The general structure of synthesized polymers has a heterogeneous thickness that suggests that they consist of several heavy blocks linked by bridges. P(q) Fitting. Having obtained this model-independent information, we attempted to fit the scattered curves using an appropriate model. The best fit is obtained with a model of noninteracting ellipsoids and generalized Gaussian coils implemented in the SASFit software.39 Attempts to fit the data to a micelle or hard-sphere model failed. Figure 13 shows the scattering curve and the fitting function for the conjugate II. One can see that the whole scattering curve from the nanoparticle can be regarded as a sum of the scattering curves of two objects: an ellipsoid with the semimajor and semiminor axes of 12.1 and 4.6 nm and a Gaussian coil with a radius of gyration Rg of about 1.4 nm. The excluded volume exponent ν = 0.52 is very close to the one predicted for ideal Gaussian chain (0.5). We thus have another direct proof of a pearlnecklace structure for the conjugates. DLS and SLS. Dynamic and static light scattering was performed to determine the average size, molecular weight and aggregation number of the nanoparticles. DLS/SLS experi-

Table 2. Molecular and Conformational Parameters of the Conjugates Determined by SAXS and DLS/SLS Methodsa sample

R0g,b nm

I I-Dox II II-Dox III IV IV-Dox V VI VI-Dox VII

3.2 2.9 5.8 5.4 8.8 6.1 6.7 8.1 6.3 8.4 10.2

R

b g.cs, nm

0.89 1.12 2.98 2.73

R0h,c nm 4.5 4.4

A2 10−8,d mol cm3/g2 143 6.9 −1.2

11.5 2.93 2.60 2.90 2.94 2.63 2.85

10.5 17.5 18.9 14.6, 15.4 26.9

5.6 −3.5 5.6 2 −1.7 14

Naggrd 1 1 6.8 5.2 8 9 10 20 14 13 32

a

If the parameters for pH 5.0 and 7.2 are different, they are presented in the same cell. bBy SAXS. cBy DLS. dBy SLS. 2601


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The DLS experiment gives an Rh value of 26.9 nm for conjugate VII (Table 2) which is consistent with the FCS experiment that shows 22 nm for the same conjugate (Figure 3a). The difference between the two values may be explained by the difference in concentrations. The highest concentration measured by FCS was 2.8 × 10−4 g·mL−1, whereas the lowest concentration measured by DLS was almost four time higher, namely, 10 × 10−4 g·mL−1. SANS. For the final proof of cylindrical symmetry, we exploited SANS, in which contrast variation can be used to investigate various structural details of the particles. Through a variation of scattering by selective substitution of hydrogen for deuterium, detailed structures can be visualized.48 This technique has been used in the past for copolymers, polymercoated particles, micelles and other self-assembled structures.12,49−55 For this reason, we used a partially deuterated polymer (Figure 1, Table 1). Two types of solutions were tested: (i) solutions in 100% D2O, where scattering comes from both, the polymer and cholesterol, and (ii) solutions in 56% D2O and 44% H2O. In the latter case, the scattering length density of the deuterated polymer is almost matched by D2O/ H2O solvent mixture and most of the scattering is due to cholesterol. Figure S2 (Supporting Information) shows the SANS curve of the polymer in 100% D2O at c = 0.02 g·mL−1. The most valuable information comes from the low-q range, where one can observe a monotonic decrease of I(q) with q. The curve follows a q−4 dependence (Figure S2), indicating that the nanoparticles have a smooth surface. The curve of the polymer in the D2O/H2O mixture follows a q−1 dependence (Figure S2, Supporting Information), indicating that the cholesterol core of the nanoparticles has an elongated structure. We conclude that contrast variation studies by SANS support an anisotropic shape of nanoparticles that consist of a cholesterol core.

Figure 13. Scattered intensity Is extrapolated to infinite dilution of the conjugate II at pH 5.0. The red curve is the fitting function.

ments are widely used for these purposes.40−47 Characterization by DLS (Table 2) revealed that the nanoparticles grow with increasing cholesterol content, which is in agreement with the SAXS data. SLS experiments were used to measure the second virial coefficient and the molecular weight Mw of the nanoparticles that was further converted into an aggregation number as Naggr = Mw(nanoparticle)/Mw(conjugate). DLS and SLS showed that with increasing z, both particle size (Rh) and aggregation number grow: Rh(I) < Rh(III) < Rh(V) < Rh(VII) (Figure 14, Table 2). Other molecular parameters, such as the

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DISCUSSION Drug delivery systems, especially those designed for cancer treatment, should comply with a number of requirements, including in-depth knowledge of the physicochemical behavior of the carrier systems. The present study was performed to contribute to this knowledge. Based on all experimental data, we reconstructed the entire self-assembly behavior of HPMA copolymer conjugates in detail. FCS experiments definitively show that self-assembly occurs above a certain threshold, which is the concentration at which the first nanostructures appear in aqueous solution, similar to the CMC in amphiphiles. The driving force for self-assembly are hydrophobic interactions between the cholesterol-containing moieties. Comprehensive support for this conclusion was obtained from all sets of experiments. The main evidence for the importance of cholesterol in nanoparticle formation is their absence in cholesterol-free conjugates: I and I-Dox. Moreover, with increasing amounts of cholesterol, nanoparticle size grows. Increasing the conjugate concentration in solution above the CMC results in a step-like growth of nanoparticles, visible by FCS (Figure 3). The existence of such jumps can be explained by the formation of pearl-necklace structures that were explicitly demonstrated by SAXS results (Figure 12). By means of SANS contrast variation, we were able to reveal that a single pearl is composed of a cholesterol core and a HPMA shell. Because cholesterol itself has an anisotropic shape, it forms parallel rod-like structures inside each pearl. When cholesterol moieties are in abundance, several pearls

Figure 14. Dependence of the nanoparticle aggregation number Naggr on the cholesterol content (z) for the Dox-free conjugates (pH = 7.2).

second virial coefficient A2, clearly indicate structural changes of particles upon inclusion of Dox (Table 2). For all Doxcontaining conjugates, the second virial coefficient A2 is negative, which is consistent with the change of the slope in the plot Rg(c) observed in SAXS experiments. The Dox moieties must be in contact with water to change the sign of A2. If they were hidden in cholesterol pearls that are strongly hydrophobic, no interaction between Dox and water could occur. The SLS data imply the presence of Dox at the nanoparticle surface. 2602


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tumor drug delivery and the in vivo treatment of mouse cancer models.

interconnected by HPMA copolymer bridges appear, thus forming an anisotropic nanoparticle. Its growth takes places through increasing the pearl length and the formation of new pearls, as shown in the PDDF analysis (Figures 9 and 11). One can observe that the length of the nanoparticles increased almost 2-fold for conjugates II and VII, whereas the radius of gyration of a particle’s cross-section was nearly the same. A core−shell cylindrical structure has already been reported for similar polymers by SANS contrast variation.12 Our experiments suggest that the presence of Dox perturbes the pearl-necklace structure. At the same length of nanoparticles, Dox conjugates are generally thinner but have more uniform thickness over the length of the nanoparticles (Table 2, Figure 10). The SAXS data strongly suggest that Dox entities are not impregnated inside of a pearl but are rather uniformly distributed within nanoparticle, including its hydrophilic shell and surface (Figure 15). The negative virial coefficients

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CONCLUSION We report a rigorous investigation of the detailed structure of nanoparticles that have already been demonstrated to be successful drug delivery nanocarriers. The basic structure of the drug conjugates consists of an N-(2-hydroxypropyl)methacrylamide copolymer bearing the anticancer drug doxorubicin (Dox) bound via a pH-sensitive hydrazone bond and a defined amount of cholesterol derivative moieties differing in their hydrophobicity. Our study of dilute aqueous solutions of conjugates in phosphate buffer at pH 5.0 and 7.2, examined by fluorescence correlation spectroscopy (FCS), dynamic and static light scattering (DLS/SLS), and small-angle X-ray and neutron scattering (SAXS and SANS), prove that nanoparticles are formed by self-assembly above a certain polymer concentration. Moreover, the content of cholesterol has a strong influence on the system. We established that the presence of any amount of cholesterol results in the formation of anisotropic nanoparticles provided the concentration is sufficiently high. The results show that the size, anisotropy, and aggregation number Naggr of the nanoparticles grow as the number of cholesterol moieties increases. SAXS and SANS experiments allowed us to determine the three-dimensional structure of nanoparticles composed of HPMA copolymercholesterol conjugates. From ab initio calculations, we conclude that the most probable structure of HPMA copolymercholesterol nanoparticles is a pearl-necklace with ellipsoidal pearls mainly composed of cholesterol covered by a HPMA copolymer shell; the pearls are connected by bridges composed of hydrophilic HPMA copolymer chains. Variation in pH perturbs the structure of the nanoparticle only slightly. Using a combination of various techniques, we have unambiguously demonstrated that Dox moieties are not only embedded inside the cholesterol domain only, but are also uniformly distributed around the entire nanoparticle, including a hydrophilic HPMA copolymer shell surface. We believe that a unique combination of the rigidity of the nanoparticles, together with easy accessibility of Dox, is responsible for the high antitumor efficiency of such conjugates.

Figure 15. Suggested structure of HPMA-cholesterol-Dox nanoparticles.

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obtained by SLS for Dox-conjugates perfectly matches this conclusion. An explanation why Dox does not stay inside a hydrophobic pearl lies in the insufficient hydrophobicity of Dox itself (the free amino group is present in the daunosamine part of the molecule). We were able to prove that the presence of Dox does not change the HPMA copolymer coil conformation very much despite some excluded volume effects. The results of Dox release experiments10,56 showed good accessibility of the drug for pH-controlled hydrolytic cleavage of the hydrazone bond and Dox release from the carrier system after entering the target cells. If most of the Dox units were hidden inside of the hydrophobic core, the Dox release rate would be much slower than the one found for this nanoparticle-based system. In agreement with the findings presented here, the rate of Dox release from the nanocarrier system was only ∼10% lower than from its fully soluble polymer conjugate analogs. We believe that this is due to the presence of most Dox entities being on the surface of nanoparticles or in a hydrophilic polymer shell rather than only inside a cholesterol core. This finding, together with the enhanced rigidity of the whole polymer carrier structure, makes these particular conjugates highly efficient for

ASSOCIATED CONTENT

S Supporting Information *

Materials; Determination of hydrazide content; Determination of cholesterol content; Determination of Dox content; Determination of molecular weights; The dependence of relaxation rate Γ on q2; Scattered intensity Is for conjugate III. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +420-608720561 (S.F.); +490-4089902224 (P.K.). Fax: +420-296809410 (S.F.); +490-4089902149 (P.K.). E-mail: sfi[email protected] (S.F.); [email protected] (P.K.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Sebastian Jaksch for help with FCS measurements. We gratefully acknowledge the HASYLAB (Hamburg, 2603


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Germany) for providing synchrotron beam time. The CzechU.S. joint research program AMVIS (Grant ME09059) is gratefully acknowledged. The authors acknowledge support from the Grant Agency of the Czech Republic (Grant P108/ 12/0640) and the Grant Agency of Academy of Sciences of the Czech Republic No. IAAX00500803. M.D. thanks the MaMaSelf (Master in Materials Science Exploiting Large Scale Facilities) master program for financial support. The Laboratoire Léon Brillouin (LLB, Saclay) is also acknowledged for beam time allocation. We also thank Anna Bogomolova for help with artwork.

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