Binding of HSA to Macromolecular pHPMA Based Nanoparticles for

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Binding of HSA to Macromolecular pHPMA Based Nanoparticles for Drug Delivery: An Investigation Using Fluorescence Methods Xiaohan Zhang,† Petr Chytil,‡ Tomaś ̌ Etrych,‡ Weiwei Liu,§ Leticia Rodrigues,§ Gerhard Winter,§ Sergey K. Filippov,‡ and Christine M. Papadakis*,† †

Physik-Department, Physik weicher Materie, Technische Universität München, James-Franck-Str. 1, 85748 Garching, Germany Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, 162 06 Prague 6, Czech Republic § Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-Universität München, Butenandtstr. 5, 81377 Munich, Germany Downloaded via KAOHSIUNG MEDICAL UNIV on June 28, 2018 at 03:29:19 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Amphiphilic poly(N-(2-hydroxypropyl)methacrylamide) copolymers (pHPMA) bearing cholesterol side groups in phosphate buffer saline self-assemble into nanoparticles (NPs) which can be used as tumor-targeted drug carriers. It was previously shown by us that human serum albumin (HSA) interacts weakly with the NPs. However, the mechanism of this binding could not be resolved due to overlapping of signals from the complex system. Here, we use fluorescence labeling to distinguish the components and to characterize the binding: On the one hand, a fluorescent dye was attached to pHPMA, so that the diffusion behavior of the NPs could be studied in the presence of HSA using fluorescence lifetime correlation spectroscopy. On the other hand, quenching of the intrinsic fluorescence of HSA revealed the origin of the binding, which is mainly the complexation between HSA and cholesterol side groups. Furthermore, a binding constant was obtained.

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for delivering drugs in vivo.11 However, the behavior of these drug carriers in blood is still poorly understood: On the one hand, thousands of proteins are present in blood plasma, which may form complexes with the drug nanocarriers.12 On the other hand, blood proteins may be influenced by the drug carriers and denaturize.13 These considerations become even more critical when the NPs are formed by random copolymers: some of the hydrophobic segments may not be included in the hydrophobic core region, but are located in the outer part, which is exposed to the aqueous environment.14 For drug delivery systems based on the self-assembly of random copolymers, this aspect must be examined carefully, since the hydrophobic segments exposed to the bloodstream may induce significant protein binding,15 which can influence the functionality of the drug carrier. We recently carried out a study of NPs formed by the pHPMA bearing cholesterol side groups (pHPMA-Chol, Figure 1) using synchrotron small-angle X-ray scattering (SAXS).16 The NPs were investigated in solutions of the most abundant protein from human blood plasma, namely, human serum albumin (HSA), up to its physiological concentration (35 mg mL−1) in phosphate buffered saline (PBS) as a preliminary stage toward real blood plasma. HSA

INTRODUCTION While traditional anticancer chemotherapy may bring along side effects, such as high organ toxicity of the drug, short plasma circulation time, and nonspecific drug distribution, the use of polymer−drug conjugates has been proved to be one of the most promising approaches to improve the delivery of drugs.1 Use of the polymer−drug conjugates reduces the toxicity of the drug by site-specific targeting, highly improved pharmacokinetics of the drug and prevents it from degradation before reaching the target site. This leads to a passive accumulation of the polymer−drug conjugate within tumor tissue, which is known as the enhanced permeability and retention (EPR) effect.2 One of the extensively studied systems are the poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA) based polymer−drug conjugates, which features both sitespecific delivery and controlled drug release.3−8 We have previously investigated a cholesterol-modified pHPMA random copolymer containing the anticancer drug doxorubicin (Dox) bound by pH-sensitive spacer to the polymer carrier (pHPMAChol-Dox) in regard to its self-assembly into micelles (which will be named nanoparticles or NPs hereafter to keep the nomenclature the same as in our previous publications).9,10 Moreover, their core−shell structure in aqueous solution9 as well as the kinetics of the drug release in tumor tissue10 were studied in detail. Above a critical micellar concentration of pHPMA-Chol-Dox, nanoparticles (NPs) having ellipsoidal shape were observed, their size being optimal (20−50 nm) © XXXX American Chemical Society

Received: March 28, 2018 Revised: June 13, 2018

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DOI: 10.1021/acs.langmuir.8b01015 Langmuir XXXX, XXX, XXX−XXX

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HSA.20−25 These experiments reveal the exact location of the binding site, where the previously observed weak binding takes place, and allow us to establish for the first time a binding constant describing the complexation of HSA with the pHPMA system. Moreover, to the best of our knowledge, we report here the first quenching experiment where a polymeric quencher has been employed, which simultaneously quenches the fluorescence of HSA and self-assembles into NPs beyond its critical micelle concentration. Combining the results from FLCS and from the quenching experiment helps to elucidate the HSA/NP interaction and gives new insight into the applied fluorescence spectroscopic methods.

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MATERIALS AND METHODS

Chemicals. 1-Aminopropan-2-ol, methacryloyl chloride, 6-aminohexanoic acid, N,N′-dicyclohexylcarbodiimide, tert-butyl carbazate, trifluoroacetic acid, 4-(dimethylamino)pyridine, cholesterol, dimethyl sulfoxide, N,N-diisopropylethylamine, 2-cyanopropan-2-yl benzodithioate, 2,2′-azoisobutyronitrile (AIBN), phosphate buffered saline (PBS), human serum albumin (HSA; ≥99%, lyophilized powder), and Atto655 were purchased from Sigma-Aldrich. Alexa Fluor 633 (AF633) and its NHS ester were purchased from Life Technologies. All other chemicals and solvents were of analytical grade. The solvents were dried and purified by conventional procedures. Synthesis of Monomers. N-(2-Hydroxypropyl)methacrylamide (HPMA), 1-(tert-butoxycarbonyl)-2-(6-methacrylamido hexanoyl) hydrazine and cholest-5en-3β-yl 6-methacrylamido hexanoate were prepared as described earlier.26−28 The structure and purity were examined by 1H NMR (Bruker spectrometer, 300 MHz) and by HPLC (Shimadzu 20VP) using a C18 reverse-phase Chromolith Performance RP-18e (4.6 × 100 mm) column with photodiode array detection (Shimadzu SPD-M20A). The eluent was water−acetonitrile with a gradient of 5−95 vol % acetonitrile, 0.1% TFA, and a flow rate of 5 mL min−1. Synthesis of Copolymers. The random amphiphilic copolymer (pHPMA-Chol) (Figure 1) was prepared by RAFT polymerization of the above-mentioned monomers, followed by dithiobenzoate end group removal and deprotection of hydrazide groups as described recently.16 The fluorescently labeled amphiphilic copolymer (pHPMA-Chol*) was synthesized by reaction of AF633 NHS ester (1 mg) with hydrazide groups of pHPMA-Chol (50 mg) in methanol in the dark 16 h at rt. The fluorescently labeled copolymer was isolated by gel filtration on a Sephadex LH-20 column in methanol followed by precipitation into ethyl acetate. The characteristics of the two copolymers are summarized in Table 1.

Figure 1. Schematic structures of the copolymers containing cholesterol groups randomly distributed along the polymer chain (pHPMA-Chol) and its fluorescently labeled analogue (pHPMAChol*).

plays a crucial rule in transporting substances, e.g., lipids including cholesterol.17,18 Using SAXS, a weak interaction between HSA and the NPs was detected in pHPMA-Chol without Dox, since the scattering from the mixed solution showed deviations from the sum of the scattering from HSA and the NPs. We attributed the observed interaction to the binding of a small fraction of the HSA molecules to the NPs. When 6 wt % of Dox was conjugated in pHPMA-Chol (i.e., pHPMA-Chol-Dox), the binding of HSA was suppressed. This difference in behavior in absence and presence of Dox was confirmed using isothermal titration calorimetry (ITC). However, the mechanism of such binding could not be resolved quantitatively in terms of obtaining a binding constant between HSA and the NPs, based on our SAXS results alone, due to the following factors: (i) The small fraction of the HSA molecules which bind to the NPs is superposed by the dominating unbound ones. (ii) The scattering signals from HSA, the NP, and the HSA/NP complex overlap in the full q range and cannot be discriminated properly. Therefore, in the present study, we concentrate on a Doxfree pHPMA copolymer (i.e., pHPMA-Chol), which interacts with HSA, as observed previously.16 To be able to distinguish the NPs from HSA using fluorescence lifetime correlation spectroscopy (FLCS), we label the pHPMA copolymers using a fluorescent dye.19 To eliminate the fluorescence signal of HSA−which hampers the characterization of the labeled NPs− a bandpass filter having a pass wavelength much higher than the emission wavelength of HSA is used. This way, the diffusional behavior of the NPs is distinguished and characterized successfully in the presence of HSA. The diffusion coefficient of the NPs is found to be unchanged upon mixing with HSA, confirming that the binding to the NPs is weak, as observed previously.16 To characterize the nature of this weak binding between the NPs and HSA, fluorescence quenching experiments were carried out, exploiting the intrinsic fluorescence emission of

Table 1. Characteristics of the Copolymer Samples

sample

Mw (g·mol−1)

Đ

cholesterol content (mol %)

pHPMA-Chol pHPMA-Chol*

28 600 n.a.

1.16 n.a.

2.1 2.1

hydrazide groups content (mol %)

dye content (wt %)

5.3 n.a.

0.8

Sample Preparation for FLCS Measurements. The dye-labeled copolymer pHPMA-Chol* was dissolved in phosphate buffered saline (PBS) at 0.05 μM, which corresponds to a dye concentration of AF633 of 0.01 μM. This dye-containing PBS (PBS*) was shaken for 24 h and used subsequently to prepare solutions of polymer and HSA. The copolymer pHPMA-Chol was dissolved in PBS*, such that its resulting concentration was 70, 140, or 280 μM (the latter corresponds to 8 mg mL−1). HSA was dissolved in PBS* at concentrations of 0, 10, and 104 μM. Then, the three HSA solutions were mixed 1:1 with the three polymer solutions, respectively, resulting in mixed solutions of pHPMA-Chol and HSA, with the polymer concentration ranging from 35 to 140 μM (the latter B

DOI: 10.1021/acs.langmuir.8b01015 Langmuir XXXX, XXX, XXX−XXX

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Langmuir corresponds to 4 mg mL−1), and the HSA concentration ranging from 0 to 52 μM (the latter corresponds to 3.5 mg mL−1). All prepared solutions were protected against bleaching using aluminum films and were shaken for 24 h to ensure a homogeneous distribution of pHPMA-Chol*. Sample Preparation for Fluorescence Quenching Experiments. In the fluorescence quenching experiment, pHPMA-Chol was used to quench the intrinsic fluorescence of HSA. Mixed solutions of pHPMA-Chol and HSA were prepared in PBS. The HSA concentration was kept constant at 15 μM in all mixed solutions. The concentration of pHPMA-Chol was varied from 0 to 70 μM, which covers a wide range around the critical micelle concentration (CMC) which is ca. 5 μM as shown previously.29

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possible bleaching. The resulting spectra, which did not deviate from each other in intensity, were averaged. UV−vis absorption spectra were measured using a Specord 205 spectrophotometer (Analytik Jena AG) in the same quartz cell at room temperature. The entrance and exit slits were both set to 1 nm. The scan speed was 50 nm s−1.

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DATA ANALYSIS Analysis of the FLCS Autocorrelation Functions. The selection of the time period for calculation of the autocorrelation function was used as implemented in the software SymPhoTime64. The obtained autocorrelation functions were fitted using the following function: ÄÅ ÉÑ| l ÅÅ i ÑÑo o yz o j Å Ño τ o ÅÅexpjj− zzz − 1ÑÑÑo G(τ) = m 1 + T j Å o o j z Å ÑÑ} o ÅÅ j τtrip z o o ÑÑÖo Å k { Ç n ~ n −1 ρi 1 D ∑ 1/2 N i=0 τ i τ y z 1 + τ jjj1 + 2z z τD, i κ D, i k (1) {

METHODS

Characterization of Polymers. The content of cholesterol and hydrazide groups in pHPMA-Chol was determined by 1H-NMR and modified TNBSA assay, respectively, as described.27 The content of AF633 dye in pHPMA-Chol* was determined by UV−vis spectrophotometry using the Lambert−Beer law and molar absorption coefficient ε632 = 159 000 L mol−1 cm−1. The molecular weights (Mw) and dispersity (Đ) of pHPMA-Chol were determined with the HPLC Shimadzu system equipped with a gel permeation chromatography column (TSKgel Super SW3000, 300 × 4.6 mm; 4 μm) and the photodiode array, differential refractive index OptilabrEX, and multiangle light scattering DAWN HELEOS II (Wyatt Technology Co.) detectors using a methanol-sodium acetate buffer (0.3 M; pH 6.5) mixture (80:20 vol %; flow rate 0.3 mL min−1). Mw and Đ were calculated using the ASTRA VI software, and the refractive increment index dn/dc 0.175 mL g−1 was used. The Optilab-rEX detector enables direct determination of sample dn/dc and solvent refractive index provides 100% recovery of the injected sample from the column. Fluorescence Lifetime Correlation Spectroscopy (FLCS). FLCS measurements were performed using a confocal laser scanning microscope Olympus IX83 (Olympus Corporation, Japan) extended by the FLIM/FLCS upgrade kit and the SymPhoTime64 software (PicoQuant GmbH, Berlin, Germany). For all measurements, a Plan Apochromat 60× (NA 1.20) water immersion objective was used. The fluorescence lifetime properties were obtained using a pulsed laser head having a wavelength of 640 nm and a pulse frequency of 40 MHz. The laser was coupled into a polarization-maintaining singlemode optical fiber. The system was controlled by a PicoHarp 300 unit using a time-correlated single-photon counting (TCSPC) protocol with a time resolution per channel of 4 ps. The detection unit consisted of two hybrid PMA detectors with a quantum yield of 45% (at 500 nm). A bandpass filter at 690 ± 35 nm was mounted in front of the detector to suppress the fluorescence emission from HSA. The confocal volume was calibrated by measuring the fluorescent dye Atto655, the diffusion coefficient of which is known to be 426 ± 8 μm2 s−1.30 For measurements of the mixed solutions, a droplet of solution (70 μL) was placed on a cover glass. Each sample was measured seven times for 600 s. The autocorrelation functions were calculated by excluding the time period, in which high intensity peaks originating probably from aggregates of dyes, were present. Moreover, autocorrelation functions, which deviated strongly or which were not smooth at high correlation times, were discarded. The remaining autocorrelation functions were fitted one by one, as described below, and the resulting hydrodynamic radii were averaged. The background and detector afterpulsing were eliminated by applying a digital filter, which was generated separately using the lifetime decay pattern of the respective mixed solutions, to the autocorrelation function. Fluorescence Quenching. Fluorescence emission measurements were performed at room temperature using a Cary Eclipse fluorescence spectrometer (Varian Inc., Palo Alto, CA). The excitation wavelength was 280 nm, and emission spectra were recorded from 300 to 480 nm. The excitation and emission slits were both set to 5 nm. The solutions were mounted in a 10 mm quartz cell (Hellma Analytics). Each measurement was repeated 6 times to detect

(

)

N is the average number of fluorescent molecules inside the confocal volume. nD refers to the number of fluorescent species having different diffusion times, which is denoted as τD,i for the ith species. ρi is the fraction of the ith species, and κ the structure factor, which is the height-to-width ratio of the confocal volume. κ was determined in a reference measurement of the fluorescent dye Atto655 in water, which has a diffusion coefficient of 426 ± 8 μm2 s−1 at 25 °C.30 T and τtrip are the triplet fraction and time, respectively. Di, the diffusion coefficient of species i, is given by Di =

1/3 ω0 2 iV y with ω0 = jjj zzz π−0.5 4τD kκ{

(2)

V is the confocal volume which was determined together with κ in the reference measurement. The hydrodynamic radius RH of each type of fluorescent particles was calculated using the Stokes−Einstein relation: RH =

kBT 6πηDi

(3)

where kB is Boltzmann’s constant, T is the absolute temperature, and η is the viscosity of the solvent. Analysis of the Quenching Experiment. The lifetime of a fluorophore can be characterized experimentally using the following equation: i t − t0 yz zz + I 0 τi zz{ k

∑ Ai expjjjjj− n−1

I (t ) =

i=0

(4)

I is the fluorescence intensity. Ai denotes the amplitude of each decay, t0 is the beginning of the decay, τi is the lifetime of each decay, and I0 is the background. The amplitude average lifetime is defined as ⟨τa⟩ =

∑ aiτi

(5)

where τi denotes the lifetime of each decay mode and ai is the corresponding fraction with Σai = 1. The obtained steady-state fluorescence intensities were corrected for the inner-filter effect, which is mainly caused by the absorption of the incident light and emitted C

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Langmuir fluorescence light by the quencher. The corrected fluorescence intensity, Icorr, is given by31 Icorr = Iobs × 10((A ex + Aem) /2)

The molecular weight and dispersity of pHPMA-Chol* cannot be determined due to the presence of the dye and its interference with the laser of the multiangle light scattering detector. However, the refractive index chromatogram of pHPMA-Chol* is similar to that of pHPMA-Chol, thus we can expect no differences in Mw, Mn, and Đ, and used the same values for the calculation of molar concentrations of pHPMAChol and pHPMA-Chol*. Diffusion Behavior of the Copolymer pHPMA-Chol*. Using FLCS, a solution of the copolymer pHPMA-Chol* in PBS at a polymer concentration of 0.05 μM was measured as a reference. This polymer concentration corresponds to a dye concentration of AF633 of 0.01 μM, which is optimal for FLCS measurements. This polymer concentration is two magnitudes below the CMC, which is at ca. 5 μM based on results reported for similar copolymers;16,29 therefore, NP formation is not expected. The autocorrelation functions obtained in three repetitions of the measurement are normalized by the number of molecules, N, and are shown in Figure 2 together with the

(6)

where Iobs is the observed intensity. Aex and Aem are the absorbance of the sample at excitation and emission wavelengths, respectively. For fluorescence quenching, the decrease in fluorescence intensity is described by the Stern−Volmer equation:31 F0 = 1 + KSV[Q ] F

(7)

where F0 and F denote the steady-state fluorescence intensities in the absence and in the presence of quencher, respectively. [Q] is the concentration of the quencher, and KSV is the Stern−Volmer quenching constant, which is the association constant for complexation in case of static quenching; In case of collisional quenching, KSV is related to the bimolecular quenching rate constant kq by KSV = kqτ0

(8)

τ0 is the fluorescence lifetime in the absence of the quencher. In the case of a heterogeneous lifetime (n > 1 in eq 4), τ0 has the value of the amplitude average lifetime given in eq 5.

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RESULTS AND DISCUSSION To characterize the interaction between the pHPMA-Chol NPs and HSA, the diffusion behavior of these NPs is investigated in PBS using FLCS in dependence on the concentration of HSA in the mixed solutions. A bandpass filter is used to suppress the fluorescence of HSA, which hampers a clear distinction of the autocorrelation functions describing the diffusion of the dye. In addition, the intrinsic fluorescence of HSA is exploited in the fluorescence quenching experiment where the identical pHPMA-Chol as investigated by FLCS is used to quench the fluorescence emission of HSA. A quantitative analysis of the quenching data reveals the quenching mechanism, i.e. either a collisional or static one, which strongly depends on the type of interaction between the NPs and HSA. For the FLCS experiments, pHPMA-Chol containing 2.1 mol % cholesterol and having Mw about 30 000 g mol−1 and low dispersity (see Table 1 for characteristics) was chosen according to results from our previous studies,9,16,27 together with its fluorescently labeled analogue (pHPMA-Chol*) containing 0.8 wt % of the far red dye AF633. The hydrazide groups content about 5 mol % was found as sufficient for attachment of about 10 wt % of anthracyclines or other anticancer drugs by pH-sensitive hydrazone bond.32 Moreover, the polymer carriers can be also labeled by fluorescent dyes for diagnostics purposes. Thus, the partial modification of the hydrazide groups by NHS ester of selected dye prior the attachment enables the synthesis of polymer theranostics, for example using doxorubicin and near-infrared dye.33 Usually, one label per polymer chain or even less, i.e. not all polymer chains are labeled, is enough for sufficient visualization of polymer in vitro and in vivo. The low content below 1 wt % helps to minimize possible effects of the highly charged dye structure on the polymer carrier behavior. Also here the amount of the bound dye was sufficient for the characterization of the polymer and formed NPs.

Figure 2. Autocorrelation functions (symbols) together with the fits of eq 1 (lines) from three FLCS measurements (shown in different colors) of pHPMA-Chol* in PBS. The polymer concentration is 0.05 μM, and the corresponding concentration of the dye AF633 is 0.01 μM.

fits of eq 1 using nD = 1. The curves feature two decays, which are attributed to the triplet state of the dye AF633 and the diffusion of the copolymer chains, respectively. In the range of higher correlation times (>0.1 ms), only slight variations of the curves are seen, indicating a consistency of the diffusion coefficients from the three measurements. The deviations in the range of lower correlation times (15 μM), F0/F becomes constant, indicating that the fluorescence intensity of HSA is not further quenched by the additional copolymer. To quantitatively analyze the quenching, eq 7 was fitted to the data in the linear region of F0/F, i.e. at a polymer concentration of 0−14 μM (Figure 4d). The slope of the fit gave the Stern−Volmer constant, kSV = (5.4 ± 0.3) × 103 M−1. Let us first assume that the quenching is caused by collisions between HSA and the polymer. To obtain the bimolecular quenching rate constant using eq 8, the fluorescence lifetime of free HSA (unquenched) must be a priori known. Despite the presence of three fluorophores in HSA, namely tryptophan, tyrosine and phenylalanine, the fluorescence emission from HSA is mainly due to tryptophan. The other two fluorophores hardly contribute to the overall intensity because of their very low quantum yield (phenylalanine) or because they are easily quenched (tyrosine).36 Numerous investigations have addressed the tryptophan lifetime in HSA in both free and quenched conditions.37−43 The heterogeneous lifetime of tryptophan was found in the range of 2−6 ns with double exponential decays (n = 2 in eq 4).37−41 Using eq 8, one obtains a value of kq in the order of 1012 s−1 M−1. This value is far beyond the limit of a diffusion-controlled bimolecular rate which is normally in the range of 109−1010 s−1 M−1. Furthermore, based on the diffusion coefficient of pHPMAChol*, D = 57.9 ± 7.9 μm2 s−1, which was obtained earlier using FLCS, the maximal distance which a pHPMA-Chol chain can travel within 6 ns is ca. 0.8 nm, which makes it impossible for pHPMA-Chol to diffuse to HSA and quench it through collision. Based on these discussions, the observed quenching process must be a static one. In this case, the obtained Stern− Volmer constant is equivalent to the association constant describing complexation between pHPMA-Chol and HSA, i.e., kSV = [pHPMA-Chol|HSA]/([pHPMA-Chol]·[HSA]) = (5.4 ± 0.3) × 103 M−1.

Interestingly, the observed static quenching seems to reach its saturation at high polymer concentration values (>20 μM), which is counterintuitive, since the Stern−Volmer theory predicts that the number of quenched fluorophores increases linearly with quencher concentration. This phenomenon of a hindered quenching efficiency clearly points to a loss in the number of quenching sites at high polymer concentration. As the polymer concentration goes beyond the CMC, the polymers form micelles. The majority of the cholesterol moieties are no longer exposed and accessible to HSA, but protected by the pHPMA chains. Therefore, it is reasonable that the static quenching of HSA is attributed to the complexation between cholesterol side groups and HSA, which has been previously reported using circular dichroism and fluorescence quenching methods.44,45 Considering that there are on average ca. 4 cholesterol side groups on each chain, we obtain a new association constant describing the binding of HSA to the cholesterol side groups in the linear region of the Stern−Volmer plot (Figure 4b), which is kSV = [Chol|HSA]/([Chol]·[HSA]) = (1.4 ± 0.08) × 103 M−1. Reasonably, this value lies below the one observed for HSA binding to free cholesterol, which is (2.3 ± 0.3) × 103 M−1, because the cholesterol groups in the present study are tethered to the chains. Furthermore, a weak blue shift together with a broadening of the emission peak of HSA at ca. 340 nm with increasing polymer concentration is observed (Figure 5). A blue shift of

Figure 5. Emission wavelength (black triangles) and fwhm (full width at half-maximum, blue circles) of the HSA spectrum in dependence on polymer concentration. The parameters were obtained by fitting single Gaussian peaks to the fluorescence spectra of HSA (eq S1).

the fluorescence emission is normally due to a change of the environment of the fluorophore from polar to nonpolar, which reduces the effect of solvent relaxation.46 This corresponds to a scenario where the polarity of the environment of HSA, which is mainly due to the polarity of water molecules, decreases when it forms a complex with pHPMA-Chol (Figure 6). In addition to that, the slight broadening of the emission peak of HSA (Figure 5) is another evidence of multiple HSA locations, namely, free in water, bound to cholesterol side groups on the unimers, or even in the outer shell of the NPs. Recalling the fact that the cholesterol groups are randomly distributed along the chain, the surface of the NPs is presumably rough and locally irregular. HSA can hence enter the NPs by collisions and bind to those few cholesterol moieties which are distributed across the hydrophilic shell. Nevertheless, since the changes of the peak position and the peak width are only minor, the number of HSA molecules, which enter the NPS in this manner, is presumably very low. G

DOI: 10.1021/acs.langmuir.8b01015 Langmuir XXXX, XXX, XXX−XXX

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Figure 6. Schematic sketch of the fluorescence quenching of HSA by binding to the cholesterol side groups on pHPMA-Chol below its CMC. Above the CMC, most cholesterol groups are protected in the NP core and thus free from such binding. However, it cannot be excluded that few HSA enters the NP and bind to the cholesterol groups which are randomly located in the shell.

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CONCLUSION We report an investigation of the interaction between the drug delivery NPs formed by the amphiphilic pHPMA-Chol and HSA in aqueous solution using a combination of FLCS and fluorescence quenching methods. pHPMA-Chol carrying the fluorescent dye AF633 is used as a label of the NPs. The fluorescent signal from HSA was suppressed by an optical bandpass filter which matches the emission spectrum of AF633. The mixed solutions of pHPMAChol NPs and HSA were measured by FLCS at varied concentrations. For all three polymer concentrations, i.e., 35, 70, and 140 μM, NPs having Rh around 27 nm were observed. For the three investigated polymer concentrations, it was found that Rh of the NP remained constant when HSA was added. It is unlikely that HSA forms a protein corona surrounding the NPs. A fluorescence quenching experiment was carried out where the intrinsic fluorescence of HSA was used as a probe to address the previously reported weak interaction between HSA and pHPMA-Chol NPs. The observed quenched fluorescence of HSA below the CMC of pHPMA-Chol was characterized as a static quenching process resulting from the complexation between HSA and the cholesterol side groups. The blue shift and broadening of the emission spectrum of HSA suggested a change of environmental polarity of HSA, which was explained by multiple HSA locations, i.e., free HSA, HSA bound to pHPMA-Chol, and HSA bound to cholesterol in the NP shell. These results indicate that the drug delivery using the pHPMA-based NPs is not affected by HSA, since only the unassociated unimers (free chains) are affected by HSA through complexation.

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AUTHOR INFORMATION

Corresponding Author

*Fax: +49 89 289 12 473. E-mail: [email protected]. ORCID

Tomás ̌ Etrych: 0000-0001-5908-5182 Sergey K. Filippov: 0000-0002-4253-5076 Christine M. Papadakis: 0000-0002-7098-3458 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Jiři ́ Pánek for his support in the FLCS measurement. We acknowledge funding by Deutsche Forschungsgemeinschaft (PA771/17-1) and the Czech Science Foundation (Grant No. 15-10527J) within the Memorandum of Understanding of Scientific Cooperation and the National Sustainability Program I (POLYMAT LO1507). W.W.L. thanks the Chinese Scholarship Council (CSC).

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REFERENCES

(1) Markovsky, E.; Baabur-Cohen, H.; Eldar-Boock, A.; Omer, L.; Tiram, G.; Ferber, S.; Ofek, P.; Polyak, D.; Scomparin, A.; SatchiFainaro, R. Administration, Distribution, Metabolism and Elimination of Polymer Therapeutics. J. Controlled Release 2012, 161, 446−460. (2) Cassidy, J.; Newell, D. R.; Wedge, S. R.; Cummings, J. Pharmacokinetics of High Molecular Weight Agents. Cancer Surv. 1993, 17, 315−341. (3) Duncan, R. Development of HPMA Copolymer-Anticancer Conjugates: Clinical Experience and Lessons Learnt. Adv. Drug Delivery Rev. 2009, 61, 1131−1148. (4) Duncan, R.; Vicent, M. J. Do HPMA Copolymer Conjugates Have a Future as Clinically Useful Nanomedicines? A Critical Overview Of Current Status and Future Opportunities. Adv. Drug Delivery Rev. 2010, 62, 272−282. (5) Kopeček, J.; Kopečková, P. HPMA Copolymers: Origins, Early developments, Present, and Future. Adv. Drug Delivery Rev. 2010, 62, 122−149. (6) Ulbrich, K.; Š ubr, V. Structural and Chemical Aspects of HPMA Copolymers as Drug Carriers. Adv. Drug Delivery Rev. 2010, 62, 150− 166. (7) Vicent, M. J.; Manzanaro, S.; de la Fuente, J. A.; Duncan, R. HPMA Copolymer-1,5-Diazaanthraquinone Conjugates as Novel Anticancer Therapeutics. J. Drug Target. 2004, 12, 503−515. (8) Griffiths, P. C.; Paul, A.; Apostolovic, B.; Klok, H.-A.; de Luca, E.; King, S. M.; Heenan, R. K. Conformational Consequences of Cooperative Binding of a Coiled-Coil Peptide Motif to Poly(N-(2Hydroxypropyl) Methacrylamide) HPMA Copolymers. J. Controlled Release 2011, 153, 173−179.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.8b01015. Background discrimination using lifetime decay patterns; filtering of the fluorescence of HSA using a bandpass filter; fitting of the autocorrelation functions of the mixed solutions of pHPMA-Chol copolymer and HSA; fitting of the HSA emission peak by a Gaussian (PDF) H

DOI: 10.1021/acs.langmuir.8b01015 Langmuir XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.langmuir.8b01015 Langmuir XXXX, XXX, XXX−XXX