Influencing Selectivity to Cancer Cells with Mixed Nanoparticles

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Influencing selectivity to cancer cells with mixed nanoparticles from albumin-polymer conjugates and block copolymers Yanyan Jiang, Sandy Wong, Fan Chen, Teddy Chang, Hongxu Lu, and Martina Heide Stenzel Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00698 • Publication Date (Web): 06 Mar 2017 Downloaded from http://pubs.acs.org on March 8, 2017

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Bioconjugate Chemistry

Influencing selectivity to cancer cells with mixed nanoparticles prepared from albumin-polymer conjugates and block copolymers Yanyan Jiang, Sandy Wong, Fan Chen, Ted Chang, Hongxu Lu, Martina H. Stenzel* Centre for Advanced Macromolecular Design, School of Chemistry, University of New South Wales UNSW, Kensington NSW 2052, Australia Email: [email protected]. KEYWORDS (polymer-protein conjugates, albumin, drug delivery)

ABSTRACT: Albumin based nanoparticles are widely used to delivery anti-cancer drug as they promote the accumulation of drugs in tumor sites. Nanoparticles with surface immobilized albumin are widely described in literature, though mixed nanoparticles with systematically modified ratios between albumin and PEG-based material are less common. In this work, hybrid nanoparticles were prepared by co-assembly of a PEG-based amphiphilic block copolymer together with a polymer-protein conjugate. Poly(oligo(ethylene glycol) methyl ether acrylate)-poly(ε-caprolactone) (POEGMEA-PCL) was prepared by a combination of ringopening polymerization and reversible addition fragmentation chain transfer (RAFT) polymerization while the polymer-protein conjugate was obtained by reacting poly(ε-caprolactone) with bovine serum albumin (BSA-PCL). Co-assembly of both amphiphiles at different ratios, with and without curcumin as a drug, led to hybrid nanoparticles with various amount of albumin on the particle surface. The resulting hybrid nanoparticles were similar in size (100-120 nm), but increasing the amount of albumin on the surface led to a more negative zetapotential. The cytotoxicity of the curcumin-loaded nanoparticles was examined on several cell lines. The curcumin-loaded nanoparticles with high amount of albumin led to high cytotoxicity against breast cancer cell lines (MDA-MB-231and MCF-7), which coincided with high cellular uptake. However, the cytotoxicity of the curcumin loaded nanoparticles against CHO cells and RAW264.7 cells was reduced suggesting that albumin can facilitate selectivity towards cancer cells.

In the treatment of cancer, the efficacy of chemotherapy has been limited by various side effects1 due to the disadvantages of the anticancer drugs such as non-selective toxicity, hydrophobicity in nature and rapid clearance from body.2 Cancer nanotechnology is a rapidly emerging field which is very promising for the revolution of cancer detection, diagnosis, treatment and cure.3 The polymer, which has been extensively used to fabricate nanoparticles, can be tailored in its properties to achieve the desired outcome and be easily achieved with high production yield.4-5 The usage of polymeric nanoparticles for anticancer drug delivery has demonstrated great therapeutic potential by conferring the drug delivery system some beneficial features such as improved solubility, prolonged circulation time and controlled drug release.6 Biodistribution, stability, solubility, immunogenicity, and nonspecific bioactivity of therapeutics can be altered using different polymers which are rationally designed for a particular application.7 Besides complicated chemical synthesis, nature also offers unique protein candidates that can act as versatile drug carriers. For example, serum albumin is a multi-functional protein that is able to bind and transport numerous endogenous and exogenous compounds.8 And it plays a peculiar role in certain diseases (e.g. cancer). It has been found that albumin is prone to accumulate in tumor sites, which could be ascribed to several factors. First of all, the accumulation in malignant tissue results from a leaky capillary as well as an absent or defective lymphatic drainage system. Secondly, as in growing tumour

tissue, albumin is taken up by cells as a source of amino acids and energy.9 Furthermore, the transportation of albumin from the bloodstream into the extravascular space is facilitated by a certain receptor on the endothelial wall. Transcytosis is initiated when albumin binds to a receptor, gp60 (a 60-kDa glycoprotein) and also known as albondin, on the surface of the endothelial wall.10-11 This receptor-ligand interaction causes the intracellular tail of the receptor to recruit caveolin-1, resulting in the formation of invaginations at the membrane surface that ultimately form transcytotic vesicles known as caveolae.12 The vesicle then passes through to the other side of the cell and fuses itself to the plasma membrane, releasing albumin into the extracellular space. Upon entering the tumor interstitium, the accumulation of albumin is more facilitated by SPARC, a 43 kDa unique glycoprotein with significant homology to gp60 and high binding affinity to albumin.13 Overexpression of SPARC is associated with increased tumor invasion, metastasis, and poor prognosis in multiple tumor types, and albumin-binding and subsequent uptake by tumor cells has been demonstrated in several in vitro and in vivo tumor models.10 These receptor glycoproteins provide a mediated albumin transport pathway to the subendothelial space and more importantly, even distribution in tumor site.10 Exploitation of these properties promotes albumin as an attractive candidate for half-life extension and targeted intracellular delivery,14 such as the recently reported polymer-albumin conjugate with pendant drug molecules,15 converting concepts of nature into macromolecular constructs that display attractive and often unexpected biological activities.

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Grafting the albumin molecularly on the surface of the traditional polymeric nanoparticles to replace the hydrophilic polymer block could combine the intrinsic properties of albumin to polymeric nanoparticles, resulting in nanoparticles with increased biocompatibility and biodegradability.13, 16 Several reports have already provided solid evidence that using albumin to design nanoparticles will facilitate the accumulation of the chemotherapeutical agents in the tumor site.17-18

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BHCT-PCL was used as a macroRAFT agent for the polymerization of the monomer oligo (ethylene glycol) methyl ether acrylate (OEGMEA) as the water-soluble block. The resulting block copolymer, POEGMEA-PCL, had a molecular weight of 23,000 g.mol-1 and a dispersity index (Ð = 1.35). The synthesis of MI-PCL was carried out according to the procedure in Scheme 1b using N-(2-hydroxyethyl) maleimide as initiator and tin(II) 2-ethylhexanoate as catalyst at 110 oC to afford polymers with low polydispersity (Ð = 1.11) and high maleimide group fidelity. The molecular weight Mn,GPC of MI-PCL was determined to be 8000 g.mol-1 based on linear PS standards. Although this technique led to better defined polymers it was not considered to be suitable for BHCT-PCL as the high temperature can potentially destroy the BHCT RAFT agent.20 Table 1. Summary of homopolymer and block copolymer for the synthesis of PCL nanoparticles. Sample name

MnTheo (g.mol -1 )

Mn GPC

Polymer

Conv.

BHCT-PCL

PCL50

95 %

5900

5200

1.8 5

POEGMEA -PCL

PCL50-bPOEGMEA3

35 %

22960

23500

1.3 7

MI-PCL

PCL67

67 %

7560

7890

1.1 1

(g.mol -1 )

Ð

7

Scheme 1. Formulation of the nanoparticles with different amount of albumin content on the surface of the nanoparticle

However, the effectiveness of albumin on the surface of nanoparticles has not yet been investigated and it is not clear what role the albumin density on the surface plays. In this work, we prepared mixed nanoparticles with varying amounts of albumin. We synthesized two different polymers based on the ringopening polymerization of ε-caprolactone, POEGMEA-PCL and maleimide functionalized PCL (MI-PCL). Thereafter, MIPCL was conjugated onto BSA by Michael Addition reaction to afford the BSA-PCL amphiphilic macromolecules. With the co-assembly of POEGMEA-PCL and BSA-PCL mixture at different ratios, nanoparticles with different albumin content on the surface were obtained. These nanoparticles were loaded with curcumin as a model drug to be able to investigate the influence of the albumin content on the physical properties (such as particle size, zetapotential and drug loading efficiency) of the nanoparticles. Main focus, herein, is to quantify the influence of the albumin on cellular uptake and the subsequent cytotoxicity of the drug loaded nanoparticles. Polycaprolactone (PCL) was chosen to work as the hydrophobic core due to its biodegradability and biocompatibility. Especially, its hydrophobic property makes it a credible candidate to form the drug pockets of nanoparticles.19 As listed in Table 1, two PCL polymers were synthesized via ringopening polymerization (ROP) of ε-caprolactone in different conditions. For the BHCT-PCL, the polymerization was performed under enzymatic condition at 70 oC in the presence of lipase resin and BHCT RAFT agent as an initiator (Scheme 1a). The conversion of this reaction was determined by the 1H NMR spectra revealing a PCL block with 50 repeating units.

Two polymers, POEGMEA-PCL and MI-PCL, were employed to generate a series of nanoparticles with various degrees of albumin functionalization (Scheme 1c, ESI Table S1). While POEGMEA-PCL could be directly self-assembled by dialyzing against water from a common organic solvent, here in DMSO-THF mixture, MI-PCL needed to be conjugated via Michael Addition to BSA first prior to further selfassembly. This conjugation process was studied in detail elsewhere.21 POEGMEA-PCL was introduced into the BSA-PCL solution at the desired ratio, followed by dialysis to formulate nanoparticles with 0%, 25%, 50%, and 75% to 100% albumin on the particle surface, denoted here as NP0-L(S), NP25, NP50, NP75, and NP100. Since increasing BSA content led to an increase in nanoparticle size as measured using DLS (Table 2), an additional nanoparticle was prepared from POEGMEA-PCL by dropping water very slowly into the organic solution, creating nanoparticles (NP0-L) that were similar in size to NP100 allowing direct comparison of the biological performance without having to consider nanoparticle size effects. Table 2. Physico-chemical characterization of blank and curcumin loaded nanoparticles. Before drug loading

After drug loading

Dh (nm)

PDI ζ (mV)

Dh (nm)

PDI

Drug loading ζ efficien (mV) ciency/ %

NP0-L

109.

0.15

-0.05

112

0.28

-0.4

45

NP0-S

51

0.34

-0.11

84

0.24

-0.1

45


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Bioconjugate Chemistry NP25

60

0.21

-11.7

112

0.23

-9.1

45

NP50

86

0.16

-14.4

120

0.17

-12.9

71

NP75

96

0.13

-16.1

120

0.11

-14.5

66

NP100

116

0.21

-27.8

123

0.30

-27.8

49

According to DLS (Table 2) and TEM (ESI, Fig. S4a to f) results, the size of the six nanoparticles in reveals the dependence of the size on the fraction of PCL-BSA. The nanoparticles NP0-S, NP25, NP50, NP75 and NP100 have an increasing negative zeta potential due to the increasing amount of albumin on the particle surface. The global structure and the repulsive negative charge of albumin lead to higher space occupation and chain repulsion, which could have contributed to the formation of larger nanoparticles with higher BSA content. NP0-L in contrast had a comparable particle size to NP100, but a zeta potential similar to NP0-S.

Figure 1. Top) TEM images of the six curcumin-loaded nanoparticles. (a) CNP0-L, (b) CNP0-S, (c) CNP25, (d) CNP50 (e) CNP75 and (f) CNP100. The scale bar is 200 nm. Bottom) Appearance of solutions showing the high solubility in water

A series of curcumin-loaded nanoparticles with different amount of albumin were prepared by adding curcumin into the organic phase (ESI, Table S2), followed by the addition of aqueous buffer solution. The successful encapsulation was made visible by the clear yellow solution of the curcuminloaded nanoparticles after eliminating the free curcumin and the organic solvent by dialysis (Figure 1). The presence of drug in the nanoparticles was not only visible by the yellow color, but also by the change in hydrodynamic diameter (Table 2). According to the DLS results, the hydrodynamic diameters of all six nanoparticles were enlarged after curcumin encapsulation. Interestingly, the particle size of the curcuminloaded nanoparticles still demonstrated an increasing tendency with the increase of albumin content in the nanoparticles. However, the size difference among nanoparticles has diminished and all the nanoparticles have now diameters of around 120 nm with the exception of CNP0-S. TEM images depicted in Figure 1 further confirmed the morphology and the size of all the nanoparticles. Moreover, the encapsulation of curcumin into nanoparticles did not cause any significant changes in zeta potential, suggesting that the drug is mainly localized in the core.

Albumin is widely used in clinical settings as a drug delivery system due to its potential to improve targeting while decreasing the side effects of both drugs and drug carriers.23 Previous in vitro studies have demonstrated that nanoparticles with albumin shells show enhanced cellular uptake by cancerous cell lines (A2780 and AsPC-1) while these particles have a lower affinity to healthy cell lines.17 It was proposed that plasma proteins, such as albumin are a major energy and nitrogen source for tumors and the oxygenated cancer cells switch metabolic pathways from glycolysis in favor of amino acid catabolism whose components necessary for energy generation and for the synthesis of tumor proteins, RNA and DNA, etc.24 In addition, there is strong evidence in literature that albumin transcytoses into cells via the gp60 receptor,25-26 an endothelial cell membrane 60-kDa albumin-binding protein localized in caveolae. A range of other cellular albumin binding proteins and receptors have also been identified.27 Central question in this work is therefore if there is a direct correlation between albumin content and selectivity and furthermore a selective delivery of curcumin to cancerous cells while healthy cells remain unaffected or less affected by the damaging effect of the drug.

The encapsulation efficiency of curcumin was determined using UV-VIS spectrophotometer. The obtained encapsulation efficiency of curcumin is also listed in Table 2. The drug loading efficiency is around 40% with some BSA nanoparticles displaying higher encapsulation efficiencies probably due to the presence of hydrophobic pockets in the global protein structure, which can provide additional cavities for the drug.

All the blank PCL based nanoparticles showed no toxicity in all of the examined cell lines (ESI, Figure S5). Previous studies28 showed the half-maximal inhibitory (IC50) values of free curcumin is in the range of 10 to 25 µM against different cell lines (Human leukemia (KBM-5 and Jurkat), breast (MDAMB-231), colon (HCT116) and esophageal (SEG-1) cancer cells). According to our cytotoxicity test, the IC50 value of free curcumin against several cell lines MDA-MB-231, MCF7, CHO and RAW264.7 is 31.37 µM, 37.5 µM, 18.78 µM and 6.8 µM, respectively (Figure 2), highlighting the high toxicity of curcumin towards non-tumor cells (CHO) and macrophages (RAW264.7).

22-23



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are desired to ensure that the fluorescence is stable under these conditions. And at the same time, the cell number in the scope especially in the red circle area has decreased after 2 h, indicating the anticancer activity of the nanoparticle.

Figure 2. IC50 of all the curcumin PCL particles with different ratios of POEGMEA-PCL and BSA-PCL against breast carcinoma cell line ((a) MDA-MB-231and (b) MCF-7) and healthy cell line ((c) CHO and (d) RAW264.7) for 48 h, and the mean ± standard deviations are shown, n=4.

Thereafter, all the curcumin-loaded PCL nanoparticles CNP0L, CNP25, CNP50, CNP75, CNP100 and CNP0-S were incubated with these four cell lines to investigate the influence of the degrees of the albumin functionalization on the cytotoxicity of the payload drug and the resulting IC50 values were also presented in Figure 2. Noticeably, the anticancer activity of the curcumin-loaded nanoparticles against MDA-MB-231 and MCF-7 cancer cells was improved as the BSA fraction on the particle surface increased (Figure 2a and b). Compared to free curcumin, all the nanoparticles led to an enhancement of the cytotoxicity against cancerous cell lines. In contrast, as shown in Figure 2c and d, CHO and RAW264.7 showed a decrease of drug activity with increasing albumin content. This effect is particularly obvious with RAW264.7 where all curcumin-loaded nanoparticles resulted in a lower toxicity than the drug alone. It should be noted here that there is no measurable influence by the particles size. Nanoparticles with higher albumin content have similar nanoparticle sizes (Table 2), while the PEGMEA nanoparticles, CNP0-L and CNP0-S, despite having different sizes, show similar behavior. The key to the different cytotoxicity lies in the difference in cellular uptake of the nanoparticles. Therefore, the curcuminloaded nanoparticles in live cells were monitored by confocal microscopy. The fluorescence behavior of curcumin enables not only tracking of the drug in the cell, but can also help identifying the environment of the drug. Curcumin, encapsulated in the nanoparticles, shows strong fluorescence but the fluorescence intensity declines when the drug is released (ESI, Fig. S6). In Figure 3, the fluorescence of the nanoparticles has good overlap with the image of the cells, suggesting that CNP75 nanoparticles have been successfully internalized by MCF-7 cells, followed by the release of curcumin inside the cell. As time elapses, curcumin is released from the nanoparticle and its fluorescence becomes weaker. It is plausible that the decline in fluorescence may be the results of fluorescent quenching by light irradiation and therefore appropriate tests

Figure 3. Confocal microphotographs of live MCF-7 cells treated by CNP75 at 37 oC for 2 h before observation The time labeled in the picture stands for the time elapse during observation. Green color indicates the presence of curcumin in the nanoparticle CNP75. When the curcumin releases out from the nanoparticle, it will lose the fluorescence. The red circle shows the decreased cells. Scale bar is 100 µm.

According to the aforementioned results, we hypothesize that BSA containing nanoparticles, which caused higher drug cytotoxicity, were more efficiently endocytosed by cancer cells. In addition, the amount of albumin on the surface of nanoparticles may have a close relation to the cellular uptake. The cellular uptake in MDA-MB-231 cells was therefore studied in details using confocal fluorescent microscopy and flow cytometry. The curcumin-loaded nanoparticles exhibited green fluorescence at the excitation wavelength of 420 nm and the cell lysosomes were stained with LysoTracker Red DND-99 (red). The fluorescence intensity of each nanoparticle was normalized.


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Bioconjugate Chemistry Important is that albumin needs to be present in its native state in order to be recognized by this receptor. This is in contrast to gp18 and gp30 receptors that preferably interact with denatured albumin and not intact albumin. While these receptors can be found on many healthy cell lines such as macrophages and fibroblasts, the conformation of albumin in our experiment was not compromised as evidenced by circular dichroism analysis. The structural integrity of albumin is crucial to target albumin receptors as denatured albumin has a higher affinity to scavenger receptors, which are mainly expressed on macrophages.36-37

Figure 4. Confocal microphotographs of MDA-MB-231 cell line after incubated in the curcumin nanoparticles at 37 oC for 2 h. Green color of the nanoparticles comes from curcumin. Lysosomes (Red) were stained with LysoTracker Red DND-99. Scale bar is 50 µm.

Increased fluorescence intensity indicative of higher accumulation of nanoparticles in MDA-MB-231 cells can indeed be observed with increasing albumin content (Figure 4, ESI Fig. S7, S8). In addition, there is no significant difference of fluorescence intensity between CNP0-L and CNP0-S, which suggests that the particle size exerts no obvious influence on the cellular uptake in this system. The cellular uptake of these PCL based nanoparticles into MDA-MB-231, MCF-7, CHO and RAW264.7 cells were further quantified using flow cytometry after 3 h incubation (ESI, Fig S9– S13). The fluorescence intensity of each sample was normalized before adding to the cells. Figure 5 summarized the percentage of cells that have internalized nanoparticles in dependency of the albumin content. As for the MDA-MB-231 and MCF-7 cells, the cellular uptake proportion of the nanoparticles correlates with the BSA amount on the surface, which confirms the results obtained with confocal microscopy. Moreover, there is a clear decrease in cellular uptake by CHO and RAW264.7 (Figure 5, Figure S12 and S13). The difference in cellular uptake, that seemed to be a key to explain the difference in toxicity in Figure 2, can be explained considering the unique interaction of albumin with various cell receptors.26, 29 Albumin receptors include gp18, gp30, gp60, calreticulin, hnRNPs as well as SPARC receptors, which play a pivotal role in transcytosis or albumin recycling. In particular SPARC has always been cited as one of the reasons for the enhanced delivery of drugs when using albumin as delivery vehicles as these receptors are often overexpressed in tumor tissue,12, 30-31 although there is not always a clear correlation between SPARC expression and activity of albumin-delivered drugs.32 The glycoprotein gp60, also coined albondin, is localized in the caveolae and can be widely found on various cells in particular on the plasma membrane.33 Albumin binds and clusters gp60 receptors resulting in caveolae-mediated endocytosis, which is the main point of entry into most cells.34 There does not seem to be any evidence that the gp60 receptor is overexpressed in cancer cells and it may not be the target for albumin nanoparticles.35

Other albumin receptors such as calreticulin and hnRNPs are overexpressed on many cancer cell lines.38-39 The interaction of native albumin with hnRNPs on MCF-7 cells has been observed earlier although it is not clear if this receptor is responsible for the enhanced albumin endocytosis.40 In this work, we investigated the endocytosis pathway of the curcumin-loaded nanoparticles using various endocytosis inhibitors (chlorpromazine, amiloride and filipin). It is found that the chlorpromazine inhibited the uptaking of nanoparticles while amiloride and filipin showed no inhibitory effect (Fig. S14). The inhibitory effect from chlorpromazine decreased with the increase of BSA content of the nanoparticle: there was even no inhibition to CNP100 which has 100% albumin on the particle surface. This result indicates the albumin receptors have important influence on the endocytosis pathways of albumin-involved nanoparticles. Considering all the different interactions of albumin with various cell surface receptors, it is not clear which mechanism is responsible for the enhanced uptake of albumin-based nanoparticles by cancer cell lines compared to the macrophage cell line RAW 264.7. The selectivity towards carcinoma cell lines compared to CHO cells can most likely be found in the differences of cellular uptake of albumin clathrin-independent endocytic mechanisms (RhoA-dependent).36

Figure 5. Flow cytometry analysis of cell uptake of the six curcumin loaded nanoparticles into MDA-MB-231, MCF-7, CHO and RAW264.7 cells after 3 h. The mean ± standard deviations are shown for n=3. CONCLUSIONS

In this work, we have investigated the influence of the albumin content conjugated on the surface of the nanoparticles on



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the accumulation and the subsequent cytotoxicity to different cell lines. The increase of the albumin content decreased the zeta potential of the blank nanoparticles but has no evident effect on the size of the corresponding curcumin loaded nanoparticles. More importantly, the high amount of albumin in the curcumin-loaded nanoparticles could result in more effective cellular uptake and consequently caused high cytotoxicity against breast cancer cell lines (MDA-MB-231and MCF-7). However, the presence of albumin lowered the accumulation of the nanoparticles in CHO cells and RAW264.7 cells so as to reduce the cytotoxicity. As a conclusion, the presence of not denatured albumin on the surface of the drug carries can enhance the selectivity of albumin towards cancer cells.

ASSOCIATED CONTENT Materials and experimental procedures, TEM analysis of blank nanoparticles, cell viability data of blank nanoparticles, fluorescent analysis of curcumin, uptake studies by confocal fluorescent microscopy and cell uptake studies by flow cytometry (raw data) This material is available free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION Corresponding Author * [email protected]

Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

Funding Sources The authors would like to thank the Australian Research Council (ARC DP140100240) for funding.

ACKNOWLEDGMENT The authors also would like to thank the Mark Wainwright analytical centre for support.

REFERENCES

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Influencing Selectivity to Cancer Cells with Mixed Nanoparticles

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