Surface Chemistry Effects on the Cellular Uptake of Biocompatible

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Nanoparticle-Cell Interactions: Surface Chemistry Effects on the Cellular Uptake of Biocompatible Block Copolymer Assemblies carlos eduardo castro, Caroline Arana da Silva Ribeiro, Alex C. Alavarse, Lindomar J. C. Albuquerque, Maria Cristina Carlan da Silva, Eliézer Jager, František Surman, Vanessa Schmidt, Cristiano Giacomelli, and Fernando Carlos Giacomelli Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b04040 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018

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Langmuir

Nanoparticle-Cell Interactions: Surface Chemistry Effects on the Cellular Uptake of Biocompatible Block Copolymer Assemblies

Carlos E. de Castro, † Caroline A. S. Ribeiro, † Alex C. Alavarse, † Lindomar J. C. Albuquerque, † Maria C.C. da Silva, † Eliézer Jäger,ϕ František Surman,ϕ Vanessa Schmidt,§ Cristiano Giacomelli,§ and Fernando C. Giacomelli†*

†

Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André,

Brazil. ϕ

Institute of Macromolecular Chemistry v.v.i., Academy of Sciences of the Czech

Republic, Heyrovsky Sq. 2, 162 06, Prague 6, Czech Republic. §

Departamento de Química, Universidade Federal de Santa Maria, 97105-900 Santa

Maria - RS, Brazil.

* Corresponding Author:

Fernando Carlos Giacomelli e-mail. [email protected]

ACS Paragon Plus Environment

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Abstract

The development of nanovehicles for intracellular drug delivery is strongly bound to the understating and control of nanoparticles cellular uptake process, which in turn is governed by surface chemistry. In this study, we explored the synthesis, characterization and cellular uptake of block copolymer assemblies consisting of a pH-responsive poly[2-(diisopropylamino)ethyl methacrylate] (PDPA) core stabilized by three different biocompatible hydrophilic shells (a zwitterionic type

poly(2-methacryloyloxyethyl

phosphorylcholine) (PMPC) layer, a highly hydrated poly(ethylene oxide) (PEO) layer with stealth effect, and an also proven non-toxic and non-immunogenic poly(N-(2hydroxypropyl)methacrylamide) (PHPMA) layer). All particles had a spherical coreshell structure. The largest particles with the thickest hydrophilic stabilizing shell obtained from PMPC40-b-PDPA70 were internalized to higher level than those smaller in size and stabilized by PEO or PHPMA and produced from PEO122-b-PDPA43 or PHPMA64-b-PDPA72, respectively. Such a behavior was confirmed among different cell lines, with assemblies being internalized to a higher degree in cancer (HeLa) as compared to healthy (Telo-RF) cells. This fact was mainly attributed to the stronger binding of PMPC to cell membranes. Therefore, cellular uptake of nanoparticles at the sub-100 nm size range may be chiefly governed by the chemical nature of the stabilizing layer rather than particles size and/or shell thickness.


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Introduction

Nano-sized soft matter assemblies are exciting platforms to overcome limitations of various methodologies in delivering active molecules to specific damaged sites.1 This is particularly interesting for the remediation of solid tumors in which sub-100 nm size particles are preferably accumulated due to a combination of a generally leaky tumor microvasculature and a missing or tight lymphatic capillary system (EPR effect).2 In such way, the use of pH-responsive nano-assemblies has great potential thanks to their ability to change physicochemical properties in distinct pH environments. Extracellular pH in solid tumors is usually acidic (ranging from pH 5.7 to 7.2)3 compared to normal tissues and blood (pH 7.4), thereby enabling pH-triggered disassembling of nanoparticles holding a pKa in such a narrow window (6.5-7.4). This is for instance the case of the polymer PDPA (poly[2-(diisopropylamino)ethyl methacrylate]) which has pKa ~ 6.8.4 Moving beyond the step of tumor accumulation, the enhanced cell internalization of nanoparticles is of due relevance since the cytosolic delivery usually potentialize the effect of anticancer drugs.5 The cellular uptake of polymer assemblies is commonly controlled by a complex process named endocytosis. The endocytic pathway involves firstly the interaction of nanomaterials with the cell membrane. This process is governed by interfacial forces typically involving electrostatic, van der Waals, depletion, hydration and hydrophobic effects. Accordingly, the endocytosis of nanoparticles is strongly dependent on the structural features and chemical composition of the assemblies. Indeed, the most relevant parameters governing cellular uptake of nanoparticles have been recently reviewed in relation to both experimental6 and theoretical-computational investigations.7 In this context, particle size has significant


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implications on cellular uptake. The endocytosis mediated by clathrin or caveolin are the main pathways of internalization for sub-100 nm particles8 with minimum and optimum sizes been suggested,9 although the ideal size for endocytosis is very hard to be predicted.7 The process is also influenced by the shape and charge where cells usually endocytose more efficiently positively charged nanoparticles due to the higher affinity of cationic species for the negatively charged cell surface.10 Regarding the shape, its influence was evidenced either in soft11 or hard12 particles where elongated assemblies are usually internalized less efficiently than the spherical counterparts as a result of longer membrane wrapping time required for elongated particles.6 Despite of that, computational works have suggested that spherocylinders can be internalized more efficiently than spheres because the previous have a smaller mean curvature.7 Hence, the shape influence still deserves further investigations. Furthermore, the surface characteristics of the assemblies affect the uptake process because it is what the cells will see. In this framework, the paramount requisite of nanoparticles for biomedical applications is the colloidal stability and thus, they are usually coated with a hydrophilic brush that generate steric forces preventing (or reducing) protein adsorption. Indeed, we have recently observed that high chain density13 and lengthy hydrophilic stabilizing shells14 substantially reduce protein fouling. However, we also found out that there should be a compromise between protein adsorption and cellular uptake since the latter correlates inversely with the shell thickness.15 These experimental evidences agree well with conclusions based on computer simulations.16 We have also evidenced that cellular uptake depends on the chemical nature of the core where nanoparticles comprising a highly hydrophobic inner compartment are more efficiently internalized.15 Truly, despite their low stability, hydrophobic nanoparticles normally exhibit enhanced cellular uptake17,18 due to the hydrophobic interactions with the cell membrane.


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Taking into account these issues and the previous findings, we moved forward and investigated the effects of surface chemistry on the cellular uptake behavior of polymeric micelles. We are confident that not only the length but also the chemical nature and the surface chain density of the hydrophilic shells influence the uptake efficiency. Normally, colloidal stability of nanoparticles is achieved by coating them with non-ionic and zwitterionic water-soluble polymers embracing for instance poly(ethyleneoxide) (PEO), poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA). Accordingly, nano-sized copolymer nanoparticles (NPs) have been produced through the self-assembly of PEO122-b-PDPA43, PMPC40-b-PDPA70 and PHPMA64-b-PDPA42 block copolymers. Since the PDPA block is insoluble above pKa ~ 6.8 due to the deprotonation of its tertiary amine groups, the procedure yielded to manufactured PDPA-core particles with distinct structural features and stabilizing water soluble shells of specific physicochemical characteristics. The structural features of the produced nanoparticles were determined via scattering techniques and imaging, and cellular uptake and intracellular localization in Telo-RF and HeLa cells were further investigated by fluorescence microscopy and flow cytometry analysis. The experimental data were examined and discussed based on the surface chain density, length and chemical nature of the stabilizing shell of the assemblies. The results can contribute to a guideline in the rational design of drug nanocarriers towards intracellular delivery of active molecules.


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Materials and Methods Samples and Chemicals The block copolymer poly(ethylene oxide)122-b-poly[2-(diisopropylamino)-ethyl methacrylate]43 was synthesized following the protocols described elsewhere and with required adaptions.19 (OH)2-poly[2-(methacryloyloxy)ethyl phosphorylcholine]40-bpoly[2-(diisopropylamino)-ethyl methacrylate]70 was synthesized following a procedure adapted from Ma et al.20 using 1-O-(2’-bromo-2’-methylpropionoyl)-2,3-rac-glycerol ((OH)2-SK-Br) as initiator and MPC and DPA monomers purchased from Aldrich and Sp2 respectively. The synthesis of poly(N-(2-hydroxypropyl)methacrylamide)64-b(2(diisopropylamino)ethylmethacrylate)72 was also performed via RAFT polymerization where the poly(N-(2-hydroxypropyl)methacrylamide) block was synthesized and used as a macro chain transfer agent (macroCTA) and the second block was subsequently grown from the macroCTA. These procedures were recently described in details.21 The molecular structure and molecular characteristics of the investigated block copolymers are given respectively in Figure 1 and Table 1. (A)

(B)

(C)

Figure 1. Molecular structure of PEO122-b-PDPA43 (A), PMPC40-b-PDPA70 (B) and PHPMA64-b-PDPA72 (C).


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Table 1. Molecular characteristics of the block copolymers. Polymer

Mn, GPC (g.mol-1)

Mw / Mn ,GPC

φPDPA

HPMA64-b-PDPA72

24500

1.21

0.63

PEO122-b-PDPA43

14540

1.20

0.63

PMPC40-b-PDPA70

26700

1.29

0.56

In Table 1 the subscripts refer to the mean degree of polymerization of each block. The number of PDPA repeating units in the PEO122-b-PDPA43 block copolymer was intentionally chosen to be smaller in order to keep in all the polymer samples a similar volumetric fraction of the hydrophobic PDPA segment (φPDPA) and at the same time a PEO-stabilizing shell of ~ 5000 g.mol-1 required to balance protein fouling and cellular uptake as we have recently demonstrated.14,15 The solvents and coumarin-6 were of analytical grade purchased from Sigma-Aldrich. The water was pretreated with Milli-Q® Plus System (Millipore Corporation).

Preparation of Coumarin-6 Labelled Nanoparticles The polymeric micelles were produced by nanoprecipitation from stock polymer/probe organic solutions prepared in ethanol. Accordingly, 5.0 mg of the solid copolymer samples along to 0.01 mg of coumarin-6 (0.2 % w/w) were dissolved into 2 mL of the organic solvent and afterwards dropwise added into 5 mL of phosphatebuffered saline (PBS) solution. The organic solvent was further removed by evaporation. This protocol is frequently used by us and other technical details can be found elsewhere.15 It is worth highlighting that by using such very low probe feeding (0.2% w/w), the loading efficiency was at least 98% meaning that the nanoparticles


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contain the same amount of loaded fluorescent probe within reasonable experimental error (± 2%). The loading efficiency was determined as described elsewhere.22

Characterization of the Coumarin-6 Labelled Nanoparticles Light Scattering: Dynamic light scattering (DLS) and static light scattering (SLS) measurements were performed using an ALV/CGS-3 platform based goniometer system (ALV GmbH). The autocorrelation functions (DLS) were collected using the ALV Correlator Control software and further analyzed using the algorithm REPES.23 The hydrodynamic radius (RH) of the nanoparticles was determined by using the straightforward Stokes-Einstein relation with D = τ -1q-2:

kB T q 2 RH = τ 6πη

(1)

being kB the Boltzmann constant, T the absolute temperature, q the scattering vector, η the viscosity of the solvent and τ the mean relaxation time. The polydispersity indexes were determined via the Cumulant analysis24 of the autocorrelation functions monitored at 90o as:

ln g1 (t ) = ln C - Γ t +

µ2 2 t .... 2

(2)

where C is the amplitude of the autocorrelation function and Γ is the relaxation frequency (τ −1). The parameter µ2 (second-order cumulant) was used to compute the polydispersity index of the samples (PDI = µ 2 /Γ 2).


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In the SLS mode, the light scattering intensity was monitored at scattering angles (θ ) ranging from 30 to 150°. The molecular weight (Mw(NPs)) and the radius of gyration (RG) of the pH-responsive assemblies were determined using the Berry approach as it provides a better approximation of the form factor for large particles (RG > 50 nm).

 Kc   Rθ

  

1/ 2

 1 = M  w ( NPs )

1/2

2 2   [1 + R G q ]  6 

(3)

the concentration c is given in mg.mL-1 and K is the optical constant expressed by:

 dn  4π n    dc  K= N Aλ4 2

2

2

(4)

Rθ (Rayleigh ratio) is the normalized scattered intensity (toluene was used as standard solvent), n is the refractive index of the solvent, NA is the Avogadro’s number and dn/dc is the refractive index increment determined using a BI-DNDC Brookhaven differential refractometer. The dn/dc in PBS for PEO122-b-PDPA43, PMPC40-b-PDPA70 and HPMA64-b-PDPA72 were determined respectively as 0.160, 0.133 and 0.143 mL.g-1. The electrophoretic Light Scattering (ELS) measurements were performed using a Zetasizer Nano-ZS ZEN3600 instrument (Malvern Instruments, UK). The measured values of electrophoretic mobility (UE) were converted to values of ζ-potential (mV) through the Henry’s equation: UE =

2 ε ζ f(ka) 3η

(5)

being ε the dielectric constant of the medium and η its viscosity. The Henry’s function f(ka) was calculated through the Smoluchowski approximation f(ka) = 1.5. All the light scattering measurements were taken at 37 ± 1 °C.


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Atomic Force Microscopy (AFM): The AFM observations were performed in contact mode using an Agilent 5500 AFM/SPM microscope. The setup used to acquire the images is described in details elsewhere.15 The samples were deposited onto silicon wafers followed by drying overnight at room temperature. The images were autoflattened and analyzed using the WSXM software.25

Biological Assays Cell Culture. Telomerase immortalized rhesus fibroblasts (Telo-RF) (donated by Princeton University, USA) and HeLa cells (donated by Universidade Federal do Rio de Janeiro, Brazil) were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium), supplemented with 10% fetal bovine serum and antibiotic solutions (penicillin 10,000 U.mL−1 and streptomycin 10,000 µg.mL−1) at 37 ºC in CO2 atmosphere.

Cell Viability. The cytotoxicity of the nanoparticles was evaluated in both cell lines. The cells were seeded into 96-well plates at density of 1x104 cells/well growth for 24 h and afterwards treated with 100 µL of DMEM plus 100 µL of solutions containing varying concentrations of nanoparticles. The cells were incubated for 24 h at 37 oC, washed with PBS and subjected to MTT assay. Briefly, 50 µL of MTT (3-(4,5dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide) reagent solution (0.3 mg.mL-1) was added to each well for 2 h at 37°C in CO2 atmosphere. The medium was aspirated and violet crystals of formazan generated by living cells at each well were dissolved in 150 µL of dimethyl sulfoxide (DMSO). The absorbance at 570 nm was measured using a Synergy microplate reader. The relative cell viability (%) was determined by comparing the absorbance at 570 nm with control wells containing untreated cells (100%).


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Evaluation of Hemolytic Effects. Complementary to MTT, hemolysis assays were conducted to evaluate the effect of the nanoparticles in blood and components. Initially, blood sample was donated by one healthy individual and collected into vacutainer tubes® (BD Bioscience) containing sodium citrate to minimize clot formation. The anticoagulated blood was then centrifuged (3500 rpm - 5 min) in order to remove plasma and the red blood cells (RBCs) were washed three times using 500 µL of PBS (pH 7.4). The stock hematocrit solution (Ht) was kept at 10 oC. Afterwards, varying concentrations of nanoparticle solutions (980 µL) were incubated for 2 h with 20 µL of the stock Ht solution at 37 oC. The negative and positive controls were respectively physiological saline (PBS pH 7.4) and Triton 2% solutions. Finally, the solutions were centrifuged (2000 rpm - 10 min) and subsequently the absorbance of released hemoglobin from damaged erythrocytes was quantified by transferring 150 µL of supernatant to a 96-well plate. The absorbance was measured at 540 nm using a microplate reader. The hemolysis (%) was calculated by using the straightforward equation: hemolysis (%) =

Abs sample − Abs negative control Abs positive control − Abs negative control

x 100

(6)

The use of human blood sample in this study was approved by the Universidade Federal do ABC under protocol 66527417.2.0000.5594.

Fluorescence Microscopy. Cellular uptake was qualitatively examined by fluorescence microscopy. The Telo-RF and HeLa cells were seeded on glass coverslips at a density of 70,000 cells/well in the cell culture medium and incubated at 37 ºC in CO2 atmosphere for 24 h. Next day, the cell culture medium was replaced by 360 µL of fresh DMEM and 90 µL of solutions containing 1 mg.mL-1 coumarin-6 labelled


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nanoparticles. After 2 h incubation time the cells were washed three times with PBS buffer and fixed with 4% paraformaldehyde for 10 minutes. Finally, the fixed cells were stained with Hoechst Stain solution (30 µL Hoechst 1:1000) for 15 min to stain the cell nuclei. The coumarin-6 is displayed in green and Hoescht stained nuclei appear blue. Images were acquired on a widefield Leica DMI 6000 B microscope (Leica Microsystems, Germany) coupled to an ultrafast Leica DFC365 FX digital camera (Leica Microsystems, Germany). In the acquisition of blue (Hoescht) and green (coumarin-6) the A4 (ex.: 340-380, DC 400, em.: 450-490) and L5 (ex.: 460-500, DC 505, em.: 512-542) cube filters were respectively selected.

Flow Cytometry. The technique was used to quantify the cellular uptake of the pH-responsive assemblies. The procedures were similar to the ones used for fluorescence microscopy experiments except that after 2 h incubation the cells were washed three times with PBS solution, harvested by trypsinization and re-suspended in 200 µL PBS medium. The acquisitions (10,000 events per sample) and further data analysis were performed using respectively a BD FACS Canto II flow cytometer and the Flowing software. The statistical analysis were realized using the one-way analysis of variance (ANOVA) followed by the Bonferroni test (p-value of 0.05 or less was considered to be statistically significant).


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Results and Discussion Characterization of the Assemblies The produced block copolymer nanoparticles were characterized via scattering measurements and atomic force microscopy (AFM). The experimental results are summarized in Figure 2 and Table 2. The regularized analysis of the autocorrelation functions (A) evidenced only the presence of single populations of scattering particles (B) in all the cases. It accordingly allowed determining the average radius (RH) and polydispersity (µ2/Γ 2) of the assemblies by the Cumulant method. The values of molecular weight (Mw(NPs)), aggregation number (Nagg = Mw(NPs)/Mw(polymer)) and radius of gyration (RG) were determined from SLS. In such way, we consider the Berry method instead of the straightforward Zimm plot because it provided a better approximation of the form factor for large particles which was particularly the case of PMPC40-b-PDPA70. The particle density was calculated by using the equation 7:

d=

M w ( NPs ) 4 π N A ( RH ) 3 3


(7)

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1.0

(A)

0.8 PHPMA64-b-PDPA72

0.6 C (t)

PEO122-b-PDPA43 PMPC40-b-PDPA70

0.4

0.2

0.0 10

-4

10

-3

-2

10

10

-1

0

10

10

1

2

3

10

10

lag time (ms)

(B)

1.0

Amplitude

0.8 0.6 0.4 0.2 0.0 0.1

1

10

100

1000

10000

RH (nm) 6.4

(C)

6.0 5.8 5.6 5.4 2.0

1/2

-4

x 10 *(mol.g )

-1 1/2

6.2

(Kc/Rθ)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.8 1.6 1.4 1.2 0

1

2

3 2

4 10

5

6

7

-2

q x 10 (cm )

Figure 2. Scattering characterization of the block copolymer nanoparticles produced from ethanol according to the legend: (A) autocorrelation functions acquired at 90o; (B) respective distributions of RH and (C) Berry plots (c = 1.0 mg·mL-1, coumarin-6 feeding ratio 0.2% w/w).


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Table 2. Structural characteristics of the block copolymer nanoparticles as determined by scattering analysis. RH

Polymer

µ2/Γ2

(nm)

RG

RG / RH

Mw (NPs)

Nagg

(106 g.mol-1)

(nm)

d

ζ

(10-3 g.cm-3)

(mV)

HPMA64-b-PDPA72

19

0.11

23

1.2

2.84

116

0.16

-12

PEO122-b-PDPA43

24

0.16

30

1.2

3.32

228

0.10

-3

PMPC40-b-PDPA70

56

0.13

64

1.1

53.3

1996

0.12

-3

The smallest micellar radius (RH) relates to PHPMA64-b-PDPA72 followed by PEO122-b-PDPA43 and ultimately PMPC40-b-PDPA70. Indeed, the PMPC40-b-PDPA70 micelles are remarkably larger than the others. The behavior is interesting since the volume fraction of the core-forming block (PDPA) is essentially the same (Table 1). Nevertheless, the results clearly show well-defined but structurally different assemblies. The larger size of PMPC40-b-PDPA70 micelles can be certainly attributed to the higher molecular weight (Mw(NPs)) which is more than one order of magnitude bigger than for the other produced assemblies. The PMPC40-b-PDPA70 nanoparticles are the assembly of 1996 chains whereas the values for PHPMA64-b-PDPA72 and PEO122-b-PDPA43 are respectively 116 and 228. The RG is also accordingly larger. The RG/RH ratio provides informations regarding the shape of macromolecular aggregate. The theoretical value of RG/RH for homogeneous hard spheres is 0.779 and it increases for less dense structures (RG/RH ~ 1 for vesicles, RG/RH > 1.5 for coils and RG/RH > 2 for rod-like structures). The RG/RH values of the produced nanoparticles are in the range 1.1-1.2 thereby suggesting the formation of at least nearly-spherical aggregates. The density of the assemblies implies that they are all highly swollen by water. This is consistent with the presence of the hydrophilic stabilizing shells.


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Although the produced micelles are spherical, their structure can vary significantly and for that reason, additional dimensions were calculated. The size of the PDPA core (Rc) was determined using equation 8 and the volume occupied by each DPA repeating unit inside the micellar core (VDPA) was further estimated using equation 9 based on the determined values of Rc.  3 M w ( micelle ) wt PDPA Rc =   4 π N A d PDPA φ PDPA

VDPA =

  

1/ 3

4 π Rc3 3 N agg DPDPA

(8)

(9)

In equation 8, the parameters wtPDPA, dPDPA and φPDPA are respectively the weight fraction of PDPA in the copolymer chains, the solid-state density of PDPA and the volume fraction of PDPA within the micellar core (φPDPA = 1 because in water we assume that the PDPA chains are all located within the micellar core). In equation 9, the parameter DPDPA refers to the degree of polymerization of the PDPA block in a given polymer sample. The corona width (W) was calculated using equation 10. W = R H − Rc

(10)

Finally, the core surface area available per corona chain was determined based on the relation: Ac =

4 π Rc2 N agg

The determined values resulting from these equations are given in Table 3.


(11)

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Table 3. Structural characteristics of the nanoparticles as calculated based on scattering data. Rc

Polymer

VDPA

W

Ac

(nm)

3

2

(nm )

(nm)

(nm /chain)

HPMA64-b-PDPA72

8.9

0.35

10.1

8.58

PEO122-b-PDPA43

9.4

0.35

14.6

4.82

PMPC40-b-PDPA70

22.8

0.35

33.2

3.27

The formation of star-like micelles (radius of the nucleus - Rc < corona width W) is suggested by the data given in Table 3 although by taking into account the weight fraction of the hydrophobic PDPA block (wtPDPA ~ 0.6) the theory predicts the formation of thermodynamic stable vesicular structures (detailed discussed hereafter). The data in Table 3 evidences that the volume occupied by a single DPA repeating unit is identical regardless the block copolymer. It implies that independently of the overall structure, the compactness of the core is the same. Nevertheless, the micellar core is much larger for PMPC40-b-PDPA70 as compared to the others. Moreover, since Rc and Nagg are known, the core surface area available per chain in the micellar corona (Ac) and the chain conformation itself can be discussed. In this framework, the smaller the area available into the core surface, the more stretched configuration the hydrophilic chains will assume. In contrast to PMPC40-b-PDPA70, a less stretched conformation is expected by the values regarding PHPMA64-b-PDPA72 and PEO122-b-PDPA43. This discussion is indeed fully compatible with the values of the core surface area available per chain in micellar corona (Ac) where the smallest value was found for PMPC and the largest for PHPMA (the smaller the space, the more stretched are supposed to be the hydrophilic chains).


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Page 18 of 35 4.85 nm 4.85 Å

(A)

400nm 0.00 Å 0.00 nm 7.64 nm

(B)

400nm 0.00 nm 20.77 nm

(C)

400nm 0.00 nm

Figure 3. AFM images of PHPMA64-b-PDPA72 (A), PEO122-b-PDPA43 (B) and PMPC40-b-PDPA70 (C) polymer nanoparticles prepared by drop-casting dilute solutions onto silicon wafers.


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The scattering data were complemented by imaging as portrayed in Figure 3. The images reveal spherical or nearly spherical particles with some degree of dispersity. Taking into account that the number of probed particles is much smaller than in the scattering measurements, we have decided not to perform quantitative size analysis. Besides, the dehydration caused by solvent evaporation usually leads to discrepancies comparing the sizes reported by DLS and AFM. Nevertheless, the most important information is the undoubtedly presence of nearly spherical core-shell block copolymer micelles whereas cylindrical aggregates and/or vesicles (polymersomes) have not been detected. This was indeed expected taking into account that the assemblies were produced by using the fast nanoprecipitation protocol. The nanoprecipitation is a simple and fast method resulting in the instantaneous formation of nanoparticles. The process presumably avoids the formation of more complex structures as time is required for instance in the production or polymer vesicles (polymersomes) although the weight fraction of the hydrophobic PDPA block is high (wtPDPA ~ 0.6).

Biological Assays

Evaluation of Cytotoxicity and Hemolytic Effects

There are several reports highlighting hemolytic activity of a variety of potential therapeutic nanomaterials thereby pointing out possible adverse side-effects that can ultimately limit applications in nanomedicine.26–29 The influence of the structural features (particularly the surface chemistry) of the produced assemblies on the hemolytic behavior was investigated by incubating the nanoparticles with red blood cells and further measuring the absorbance at λ = 540 nm. The hemolytic behavior was


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evaluated by exposing red blood cells to concentrations ranging from 0 to 500 µg.mL-1 and the data are given in Figure 4. The differences in optical density of experimental samples as compared to the control were never higher than 1% regardless the concentration and whatever the system thereby demonstrating the biocompatibility of the produced entities with RBCs.30 Since the block copolymer nanoparticles did not stimulate erythrocyte hemolysis they can be considered biologically safe regarding hemolytic effects. Similarly, the MTT cytotoxicity assays reveal that Telo-RF fibroblast cells in the culture remain viable at least up to c ≤ 1000 µg.mL-1. The results evidence that the number of viable cells remain very close to 100% for the whole set of nanoparticles in the investigated concentration range. The only exception is PMPC40-bPDPA70 at the highest concentration. Nevertheless, as compared to the control, the cell viability is still greater than 70%. According to the ISO 10993-5:2009 only reduction of cell viability by more than 30% is considered a cytotoxic effect. Additionally, the MTT assays sometimes show cell viability higher than 100% (higher than the control). This may be caused for instance by proliferative effects (cells are proliferating more than the control in the presence of the nanoparticles). Moreover, sometimes the investigated compounds induce increased enzymatic activity leading also to values higher than 100%. Besides, small variations in the number of cells seeded per well due to small pipetting errors may lead to such results since the conversion of tetrazole to formazan is dependent on the number of viable cells. Nevertheless, as only mitochondrial dehydrogenase of viable cells are able to convert the yellow reagent into purple formazan, the assay is very useful to mark only the living cells and accordingly, the Figure 4 convincingly shows that the cytotoxicity of the investigated nanoparticles is negligible. The cell viability was evaluated also in HeLa cells and the behavior seems to be cell line independent (Figure S1 - Supporting Information File)


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1.0

HPMA64-b -PDPA72

(A)

PEO122-b -PDPA43

hemolysis (%)

0.8

PMPC40-b -PDPA70

0.6

0.4

0.2

0.0

140

25

200 100 50 -1 concentration (µg.mL )

Telo-RF cells control

500

(B)

120

cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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100 80 60 40 20 0

50

100

200

500

1000

-1

concentration (µg.mL )

Figure. 4 Hemolytic behavior of the nanoparticles incubated with RBCs (A) and cell viability of Telo-RF cells against varying concentrations of nanoparticles in contact for 24h (B). The results were normalized by setting the viability of the control to 100% (n ≥ 3).


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Cellular Uptake

The cellular uptake was evaluated qualitatively and quantitatively using fluorescence microscopy and flow cytometry. The qualitative data are given in Figure 5 for Telo-RF and in Figure S2 (Supporting Information File) for HeLa cells.

Coumarin-6

DAPI

Overlay

A

B

C

Figure 5. Fluorescence microscopy images of Telo-RF cells incubated for 2 h with coumarin-6 labelled PMPC40-b-PDPA70 (A) HPMA64-b-PDPA72 (B) and PEO122-bPDPA43 (C) nanoparticles.


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The cellular uptake was visualized by fluorescence microscopy in order to evaluate qualitatively the degree of internalization as well as the intracellular distribution of the polymeric assemblies. The images were obtained from DAPI channel (blue), FITC channel (green) and the overlay of the two channels. The use of coumarin6 is straightforward in cellular uptake studies of nano-sized particles for observations by fluorescence microscopy.31,32 The images reveal intensity of green fluorescence after 2 h incubation regardless the assembly. They also evidence that the cytoplasm emits considerable green fluorescence and coumarin-6 is almost absent in cell nuclei. The behavior is similar in HeLa cells (Figure S2 - Supporting Information File). This signs that a considerable amount of nanoparticles are uptaken by the cells after 2 h. Additionally, the images point out interesting and important aspects: Firstly, the evenly distribution of fluorescence in the cytoplasm ruled out the preferentially partition of the dye into the cell membrane (coumarin-6 is mostly not membrane associated). Moreover, the different degrees of internalization, as hereafter discussed, suggest that internalization is linked to the endocytosis of the nanoparticles that is the main mechanism used by the cells to capture nano-objects. Presumably, the dye cannot be easily transferred directly into the cells without the capturing of the nanoparticles otherwise similar fluorescence intensities would be monitored. Finally, at least part of coumarin-6 seems to be still associated with the particles as evidenced by partial punctate fluorescence signals. In the step further, we evaluated the uptake quantitatively via flow cytometry. The Figures 6A and 6B portray respectively the flow cytometry histograms and mean fluorescence intensity resulting from the cellular uptake of coumarin-6 labelled polymer nanoparticles after 2 h incubation in Telo-RF cells (control - autofluoresence of Telo-RF


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cells). The analogous data for HeLa cells are given in the Supporting Information File (Figure S3).

1.0

(A)

Count (a.u.)

0.8

0.6

0.4

0.2

0.0 0 10

1

2

10

3

10

4

10

10

Fluorescence Intensity Telo-RF cells - control PEO122-b-PDPA43 2500 Mean Fluorescence Intensity (MFI)

HPMA64-b-PDPA72 PMPC40-b-PDPA70

*#&

(B)

2000

*&

1500

* 1000

500

PE O 12 2 -b -P D PA 43 H PM A 64 -b -P D PA 72 PM PC 40 -b -P D PA 70

0

Te lo -R F

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 6. Quantitative flow cytometry data for coumarin-6 labelled nanoparticles uptaken by Telo-RF cells after 2 h incubation (A) and respective mean fluorescence intensity per 10 000 events (B). The results are expressed as mean ± SD (n = 3). *represents statistically significant difference compared to the control,

&

statistically significant difference

#

compared to PEO122-b-PDPA43,

represents represents

statistically significant difference compared to PHPMA64-b-PDPA72.

The untreated Telo-RF cells show only autofluoresence and then, the monitored fluorescence intensities are proportional to the amount of coumarin-6 labelled


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nanoparticles internalized by the cells. At this point, we highlight again that the amount of coumarin-6 was quantified and normalized for each nanoparticle enabling the interpretation of the cellular uptake data. It is widely accepted that a hydrophilic stabilizing shell is required in order to avoid, or at least postpone, the opsonisation of nanoparticles in the bloodstream. Amongst the alternatives, the most commonly used polymer is PEO, but others can be chosen such as those herein investigated (PMPC and PHPMA). The concept is the same where one wants the generation of stealth assemblies thereby reducing protein adsorption and ultimately delaying clearance. On the other way around, the stealth property of nanoparticles usually reduces its cellular uptake and a balance of capture and protein adsorption is often required. The current results point out that the fluorescence intensity of cells treated with coumarin-6 PMPC40-b-PDPA70 nanoparticles is about 2.5-fold and 1.7-fold higher than PEO122-b-PDPA43 and HPMA64-b-PDPA72, respectively. This accordingly means that cellular uptake of coumarin-6 loaded HPMA64-b-PDPA72 is about 1.5-fold higher than the cellular uptake of coumarin-6 PEO122-b-PDPA43 nanoparticles. Although it is accepted that particle size plays an important role in cellular uptake, the reported results shows that the largest nanoparticles are internalized to a greater extent and confirms that the capture markedly depends on other physicochemical parameters. Indeed, a considerable number of theoretical and experimental investigations converged to the conclusion that the cellular uptake of nanoparticles reaches its maximum at optimal radius in the range ~ 25-30 nm9,33–35 and it goes down for larger particles.36,37 This is claimed to be linked to a balance between the elastic energy associated with the membrane bending and the receptors diffusion kinetics. This size region is substantially far from the size monitored for PMPC40-b-PDPA70 and indeed, very close to the monitored sizes for PHPMA64-b-PDPA72 and PEO122-b-PDPA43 micelles. It advises that


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in the current investigation the degree of cellular uptake is governed by the chemical nature of the shell and not by particle size otherwise, taking into account the suggested optimal radius for cellular uptake, the PHPMA64-b-PDPA72 and PEO122-b-PDPA43 would be internalized to a greater extent compared to the PMPC40-b-PDPA70 micelles. Additionally, the longer the hydration layer the weaker the cell adhesion and cell internalization as evidenced also by us.15 However, although the PMPC40-b-PDPA70 micelles having the thickest shell, they are internalized to the highest degree implying the role of the chemical nature. The herein reported results indicate that PMPC has to be a stronger cell-binding to overwhelm the hydrophilic shell thickness and size influences. Truly, the PMPC was already suggested to have strong affinity toward specific receptors that are expressed by most cells and the evidenced higher degree of cellular uptake of the PMPC40-b-PDPA70 micelles is assisted by at least three reasons: i) favorable electrostatic interactions between the cationic choline group of PMPC and negatively charged cell membranes; ii) the ability of zwitterionic polymers to better resist protein adsorption16 and iii) the high affinity of phosphorylcholine groups for scavenger receptors that are expressed in a variety of cells.38–40 Indeed, the third reason was suggested to substantially contribute to the high degree of internalization of PMPCb-PDPA polymersomes.41 Hence, the favorable interactions between PMPC40-b-PDPA70 micelles and cell membranes may firstly enable nanoparticle-cell membrane adhesion ultimately leading to enhanced cellular uptake. Finally, although the HPMA64-bPDPA72 micelles hold a slightly more negative ζ-potential compared to the others, its degree of cellular uptake is respectively higher and smaller than PEO122-b-PDPA43 and PMPC40-b-PDPA70. If the uptake would be largely influenced by the surface charge, then one would expect smaller degree of cellular uptake for HPMA64-b-PDPA72 compared to PEO122-b-PDPA43 for instance, because negative surfaces restrict cellular


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uptake. Nevertheless, this has not been evidenced and therefore, the small differences in the overall surface charge of the assemblies are not enough to substantially influence the cellular uptake behavior. In summary, we have recently demonstrated that cellular uptake is substantially influenced by the length (or molecular weight) of PEO stabilizing shells and by the chemical nature of the hydrophobic core. At this contribution, we demonstrate that the capture is also substantially influenced by the chemical nature of the stabilizer and among the investigated shells, PMPC is the best stabilizer regarding cellular uptake. There are other experiments underway where we investigate the influence of the chemical nature on protein adsorption.

Conclusions

The production and detailed characterization of PDPA-based nanoparticles were performed and cellular uptake of the pH-responsive assemblies stabilized by different shells was evaluated by means of fluorescence microscopy and flow cytometry analysis. The whole set of block copolymers self-assembled into core-shell particles and the PMPC40-b-PDPA70 block copolymer led to the largest nanoparticles with the thickest hydrophilic stabilizing shell. Nevertheless, the PDPA-core particles stabilized by PMPC were internalized to higher extend as compared to those stabilized by PEO or HPMA. The behavior was attributed essentially to the stronger cell-binding property of PMPC to cell membranes. Similar behavior was also reported for PDPA-based polymersomes by others. The results point out that, at least at the sub-100 nm region, the chemical nature of the stabilizer seems to play a more important role in cell capturing than does the size and the thickness of the stabilizing shell which are known to also influence


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substantially the degree of cellular uptake. Finally, the assemblies are internalized to slightly higher extent in cancer (HeLa) compared to healthy (Telo-RF) cells. In both cases, however, the highest degree of internalization was evidenced for PMPC40-bPDPA70 micelles.

Associated Content

Complementary biological assays performed using HeLa cell line: cell viability by MTT, fluorescence microscopy and flow cytometry data.

Acknowledgements

This study was supported by FAPESP (Grant No. 2017/00459-4) and CNPq (400124/2016-5). F.C.G and C.E.C acknowledge fellowships granted by CNPq (Grant No. 302467/2014-9) and FAPESP (Grant No. 2015/24686-4), respectively. E.J thanks the Czech Science Foundation (grant #17-09998S). The CEM at UFABC is acknowledged for the accessibility to the Malvern light scattering equipment. The authors are grateful to A. G. O. De Freitas and P. I. R. Muraro for their assistance on polymer synthesis.

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Nanoparticle-Cell Interactions: Surface Chemistry Effects on the Cellular Uptake of Biocompatible Block Copolymer Assemblies

Carlos E. de Castro, † Caroline A. S. Ribeiro, † Alex C. Alavarse, † Lindomar J. C. Albuquerque, † Maria C.C. da Silva, † Eliézer Jäger,ϕ František Surman,ϕ Vanessa Schmidt,§ Cristiano Giacomelli,§ and Fernando C. Giacomelli†*

TOC - Graphical Abstract (For Table of Contents Use Only)


Surface Chemistry Effects on the Cellular Uptake of Biocompatible

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