Nano-Sized Albumin-Copolymer Micelles for Efficient Doxorubicin Delivery
© The Author(s) 2012
Received: 2 October 2011
Accepted: 18 November 2011
Published: 9 February 2012
We present the discovery of a nano-sized protein-derived micellar drug delivery system based on the polycationic albumin precursor protein cBSA-147. The anticancer drug doxorubicin (DOX) was efficiently encapsulated into nanosized micelles based on hydrophobic interactions with the polypeptide scaffold. These micelles revealed attractive stabilities in various physiological buffers and a wide pH range as well as very efficient uptake into A549 cells after 1 h incubation time only. In vitro cytotoxicity was five-times increased compared to free DOX also indicating efficient intracellular drug release. In addition, multiple functional groups are available for further chemical modifications. Based on the hydrophobic loading mechanism, various classical anti-cancer drugs, in principle, could be delivered even synergistically in a single micelle. Considering these aspects, this denatured albumin-based drug delivery system represents a highly attractive platform for nanomedicine approaches towards cancer therapy.
Nanomedicine has emerged as an innovative strategy for cancer therapy with great promise for clinical applications . Nanomedical approaches to drug delivery involve the development of nanoscale particles or macromolecules to improve the biological activity and pharmacokinetic profile of drug candidates . In particular, small-molecule chemotherapeutics often elicit severe side effects, such as anemia, vomiting, diarrhea, decreased immunity and alopecia, which is due to the systemic distribution of cytotoxic drugs . In recent years, a large number of natural and synthetic macromolecules as well as inorganic particles have been developed as drug delivery nanocarriers , such as liposomes [4–6], polymeric nanoparticles [7, 8], inorganic nanoparticles  as well as proteins [10–13]. The long systemic circulation times of nano-sized drugs and drug carriers and the increased vascular permeability of tumor tissue leads to an accumulation at tumor sites, which is often referred to as passive targeting or “enhanced permeability and retention effect” (EPR effect) [14, 15]. As a particular feature, such nano-sized drug delivery systems could serve as platform to integrate multiple additional functionalities such as specific targeting ligands or probes suitable for tumor imaging [16, 17]. Still, there are major challenges associated to the design of an ideal macromolecular drug delivery system [2, 3]. In order to reduce drug-unrelated side-effects in humans, biocompatibility of the entire drug delivery complex represents a key concern to achieve a suitable therapeutic window. In addition, the prevention of drug leakage during blood circulation on the one hand but achieving efficient drug release at target cells on the other hand represent crucial factors that still appear challenging to achieve. Other prerequisites include efficient permeability through vascular barriers and cell membranes, great specificity for tumor tissue, high loading of the drug cargo as well as fast degradation of the entire carrier system after delivery producing non-toxic metabolites. In view of these criteria, it is not astonishing that only few drug delivery systems have entered clinical trials yet.
Doxorubicin (DOX) represents a potent cytotoxic drug that has been applied to address a broad number of different kinds of cancers [18, 19]. It consists of an anthracycline antibiotic DNA intercalator inhibiting DNA replication, which has been successfully applied in cancer chemotherapy [18, 19]. For solubility reasons, DOX needs to be administered intravenously as hydrochloride salt, which limits its cellular uptake and cardiac toxicity due to unspecific cell uptake represents a serious side-effect . Therefore, liposome formulations such as DoxilTM containing DOX have entered the market . However, inherent limitations of liposomes such as drug leakage into the plasma and vascular capillaries still leaves room for improvement [22, 23]. In addition, dendrimers have been applied successfully for the complexation of DOX . Dendrimer mediated complexation is advantageous in terms of stability, controlled release, drug payload and reduced toxicity of the entrapped drug(s) . However, even though studies on non-covalent interactions of dendrimers with drugs suggest improved drug solubilization, low numbers of encapsulated drug molecules and limited complex stabilities represent key concerns .
Protein-based drug delivery systems have emerged recently since they consist of “natural”, non-toxic monomers, they reveal low cytotoxicity and particularly human serum albumin (HSA) is known to exhibit insignificant immunogenicity . HSA is a well-known transportation protein ubiquitous in the blood plasma to allow the delivery of hydrophobic nutrients . Utilizing its native transportation mechanism, albumin formulated cancer drugs, such as AbraxaneTM (albumin formulated paclitaxel for treating breast cancer), have been successfully introduced to the market and show higher efficiencies in patients without prominent side-effects such as allergic reactions [27–29]. However, in general, protein-based delivery platforms also bear several draw-backs such as their low membrane permeability, immunogenicity and low metabolic stability.
Herein, we disclose a novel type of denatured albumin-based copolymer that facilitates encapsulating hydrophobic drug molecules thus forming nano-sized, stable micelles that are able to efficiently pass cell membranes. Recently, protein-derived copolymers were introduced, consisting a polypeptide backbone and several grafted, hydrophilic polyethylene(oxide) (PEO) side chains (Wu et al., in preparation, [30–32]). PEO side-chains are known to reduce non-specific interactions as well as immunogenicity and antigenicity of proteins and peptides. The promising applications of such copolymers include surface patterning (Wu et al., in preparation), nanoparticles coating , gene delivery  as well as targeted delivery of hydrophobic molecules into cancer cells . Such biohybrid micelles represent a novel type of delivery platform offering great potential to effectively transport and release lipophilic drugs into cancer cells.
Albumins from bovine serum (BSA) (≥98%, Sigma Aldrich), O-(2-maleimidoethyl)-O′-methyl-polyethylene glycol 5000 (PEG-5000-MI) (>90% NMR, Aldrich), propargylamine (98%, Aldrich), maleic anhydride (≥99.0%, Fluka), glacial acetic acid (ACS 99.7%, Alfa Aesar), N-hydroxysuccinimide (98%, Aldrich), tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (98%, Alfa Aesar), Urea (98+%, Alfa Aesar), ethylenediaminetetraacetic acid (EDTA) (99%, Alfa Aesar) and TrizmaTM base (BioUltra, ≥99.8%, Sigma), doxorubicin hydrochloride salt (DOX) (≥99.0%, XingCheng ChemPhar Co. LTD, China) were used as received without further purification. Bio-Rad Bio-Gel P30 was used for desalting, vivaspin ultrafiltration tubes were purchased from GE healthcare for purification. Dulbecco’s Modified Eagle Medium (DMEM) (1X) liquid (high glucose), Fetal Bovine Serum (FBS) Standard Quality (EU approved), penicillin/streptomycin solution (100×) were purchased from PAA Laboratories GmbH, MEM [non-essential amino acids solution 10 mM (100×)] was purchased from Invitrogen.
2.2 Synthesis of N-Propargyl Maleimide
Maleic anhydride (980 mg, 10 mmol) was reacted with propargylamine (550 mg, 10 mmol) in 20 mL glacial acetic acid at room temperature (RT) overnight. Then, sodium acetate (164 mg, 2 mmol) and acetic anhydride (10 mL) were added and the reaction mixture was heated to 65°C for 2 h. After reaction, all solvent was removed under vacuum and the residue was purified by silica gel column chromatography (EtOAc:Hexane 1:3) to yield 60 mg of a white solid (yield 5%). Rf 0.56 (EtOAc/heptane, 1:1 v/v), 1H NMR (CDCl3, 300 MHz): δ 6.75 (s, 2H), 4.28 (d, 2H, J 1.8 Hz,); 2.20 (t, 1H, J 1.8 Hz); 13C NMR (CDCl3, 300 MHz): δ 169.2, 134.4, 76.9, 71.5, 26.8. MS–ESI, 136 M+.
2.3 Preparation of cBSA-147-PEO(5000)28 (2)
dcBSA-147-PEO(5000)28 (2) was prepared using a similar procedure reported in our previous paper . Briefly, cationic bovine serum albumin, cBSA-147  (1, 10 mg, 0.15 μmol) was first denatured in degassed urea-phosphate buffer (10 mL, 10 mM phosphate buffer, 5 M urea and 2 mM EDTA, pH 7.4) for 10 min, and then reducing agent TCEP (4.3 mg, 15 μmol) was added under argon atmosphere for 30 min. Subsequently, PEG-5000-MI (77 mg, 15 μmol) was added to the reaction and stir at RT for 3 h. Finally, the capping reagent N-propargyl maleimide (30 μmol) was given to the reaction and stir for another 3 h. The reaction mixture was first purified by ultrafiltration with Tris–HCl buffer (20 mM Tris, 150 mM NaCl, 2 mM EDTA, pH 7.4), and followed by further size exclusion purification using HiPrepTM SephacylTM S-100 HR gel filtration column on AKTÄ Purifier flash protein liquid chromatography with Tris–HCl buffer (20 mM Tris, 150 mM NaCl, pH 7.4). Then, the purified material was desalted and lyophilized to yield dcBSA-147-PEO(5000)28 (2) as white fluffy solid. The product was characterized by gel electrophoresis using precast NuPAGE TA 3–8% Gel (Invitrogen) in NovekTM Mini-Cell.
2.4 Preparation of dcBSA-147-PEO(5000)28 Micelle (3)
DOX hydrochloride (DOX × HCl) (4 mg, 7 mmol) was dissolved in 500 μL deionized-distilled water and mixed with 1.2 equivalents of triethylamine (1.2 μL, 8.4 mmol). The aqueous solution was extracted five times with dichloromethane. The combined organic extracts were evaporated in vacuum and 3 mg of DOX were obtained. This DOX stock solution was prepared by dissolving the obtained solid in 500 μL of DMF to achieve a total concentration of 6 mg/mL. dcBSA-147-PEO(5000)28 (2) (0.5 mg, 0.0028 μmol) in distilled-deionized water was combined with the desired molar ratios of DOX, (ratios of 1:1, 1:5, 1:10, 1:20, 1:50, 1:100) and allowed to stir overnight in the dark at RT. The reaction mixtures were transferred to 3.5 K MWCO Slide-A-Lyzer MINI Dialysis cassettes (Pierce) and dialyzed in 1 L distilled-deionized water for 24 h at 4°C; during this time period, water was exchanged 3–5 times. The amount of entrapped DOX in each molar ratio was determined by measuring the absorbance at 488 nm using Tecan M-1000 microplate reader.
2.5 Negative Staining Transmission Electron Microscopy
The morphology of dcBSA-147-PEO(5000)28-(DOX)14 micelles was characterized via TEM applying the negative staining technique. A drop of 1 mg/mL dcBSA-147-PEO(5000)28-(DOX)14, dissolved in an aqueous solution was deposited onto a hydrophilic copper grid with a carbon film for approximately 1 min and then blotted excess of the sample by filter paper. The grid was allowed to dry at RT for overnight and then stained with 1% Uranyl acetate solution for TEM investigations.
2.6 Dynamic Light Scattering
The hydrodynamic size distribution of dcBSA-147-PEO(5000)28-(DOX)14 micelles was characterized by DLS using a Malvern Nanosizer (Malvern Ltd, Malvern, UK). dcBSA-147-PEO(5000)28-(DOX)14 was prepared at 0.15 mg/mL concentration in aqueous solution and filtered through 0.2 μm microsyringe filter before measurement to avoid dust contamination. Autocorrelation functions were analyzed by cumulants method and COTIN routine to estimate hydrodynamic diameter. The hydrodynamic diameter distribution was presented as number distribution.
2.7 Characterization of Micelle Stability in Various Media
100 μL aliquots of 0.1 mg dcBSA-147-PEO(5000)28-(DOX)14 samples were dialyzed using 3.5 K MWCO Slide-A-Lyzer MINI Dialysis devices (Pierce) in 10 mM pH 3 and 5 citrate buffer and 10 mM (pH 7 and 9) and Tris buffer, respectively, for 2 days and the absorbance spectra were recorded before and after dialysis.
2.8 Cell Culture
Hela cells (human cervix carcinomic cell line) and A549 cells (carcinomic human alveolar basal epithelial cell line) were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig) and cultured in DMEM medium with high glucose supplemented and with 10% fetal bovine serum (FBS), 100 U/mL Penicillin, 0.1 mg/mL Streptomycin, 0.1 mM non-essential amino acids at 37°C in a humidified 5% CO2 incubator.
2.9 Cytotoxicity Assay
HeLa cells were plated into a white 96 well microplate at a density of 8000 cells per well and incubated overnight for attachment. After incubation, the media from each well were aspirated and exchanged with 100 μL of fresh DMEM medium and the desired amount of either free DOX or dcBSA-147-PEO(5000)28-(DOX)14 micelles. All concentrations were prepared as triplicates. After further incubation with drug molecules for 12, 24 and 48 h, cell viability was tested by Cell-titer-GloTM (Promega) cell viability assay kit according to manufactory’s instruction. Cells that were not treated with any drug were considered as blank. The IC50 values and the 50% inhibitory concentrations were obtained through GraphPad Prism 3 software.
2.10 Live Cell Imaging of Intracellular DOX in A549 Cells
A549 cells were plated onto glass cover slips and incubated for 12 h to allow cell attachment and spreading. Thereafter, 0.1 μM of either free DOX or dcBSA-147-PEO(5000)28-(DOX)14 micelles were added to the cells and incubated for one additional hour. The cells were then washed 3 times with PBS, replaced with fresh medium and incubated with 5 μg/ml of WGA-Alexa Fluor 594 conjugate (Cat # W11262, Invitrogen) for 10 min to label the cell membrane and imaged without further washing. Microscopy was performed with a Zeiss LSM 710 META laser scanning microscope fitted with Argon, HeNe543 and Diode405-30 lasers. Z-sections of chosen field were acquired with a 40× oil immersion objective (Zeiss, Germany). Excitation and emission wavelengths of 535–562 nm for DOX and of 600–620 nm for WGA-Alexa Fluor 594 detection were selected. The acquired images were processed with Zen software developed by Carl Zeiss.
2.11 Quantification of DOX Uptake
For the quantification of DOX cell uptake, images obtained from five different sections in each of the slides were analyzed using the public domain NIH ImageJ program with default settings (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). To calculate the percentage of intracellular DOX, the fluorescence intensity of DOX [green] was divided by the total fluorescence (DOX [green] and the cell membrane [red]) and multiplied by 100.
3 Results and Discussion
3.1 Preparation and Characterization of dcBSA-147-PEO(5000)28 DOX Micelles
Serum albumin transports water-insoluble lipids in the bloodstream and about six hydrophobic ligands could be transported . After denaturation, the tertiary structure of dcBSA-147-PEO(5000)28 has been destroyed but sufficient numbers of lipophilic amino acids and additional N-propargylamine are still present that could non-covalently interact with hydrophobic molecules. DOX HCl was chosen as model drug since it has been well characterized in vitro and in vivo, it has a lipophilic scaffold and emits above 500 nm thus facilitating characterization via fluorescence microscopy. DOX loading was performed according to published procedures . Briefly, DOX hydrochloride salt was first neutralized and extracted into a dichloromethane layer. The obtained non-water soluble drug was then re-dissolved in a minimum amount of dimethylformamide and added into dcBSA-147-PEO(5000)28 (2, 1 mg/mL) dissolved in aqueous solution. After overnight stirring, micelles were purified by dialysis. The purification efficiency was accessed by loading DOX micelles (3) and free DOX HCl into Bio-Gel P30 (Bio-Rad) size exclusion columns. Free DOX HCl is indicated by a red colored narrow band, which remains on top of the column, whereas sufficiently stable DOX micelles quickly moved along the column and no traces of free DOX were present on top of the column suggesting that all DOXs were associated with the copolymer micelles (Fig. 2c).
3.2 Characterization of dcBSA-147-PEO(5000)28 DOX Micelles
Summary of the loading efficiency of DOX molecules into dcBSA-147-PEO(5000)28 micelles
Ratio added DOX/(2)a
Loading efficiency (Molar ratio)b
Loading efficiency (in wt%)c
Encapsulation efficiency of DOX (%)d
0.5 ± 0.1
0.15 ± 0.03
51.6 ± 9.9
1.2 ± 0.1
0.34 ± 0.01
23.1 ± 1.0
1.8 ± 0.2
0.52 ± 0.07
17.2 ± 2.3
4.3 ± 0.8
1.22 ± 0.23
20.4 ± 3.9
8.2 ± 1.1
2.33 ± 0.31
15.5 ± 2.0
13.7 ± 2.2
3.91 ± 0.63
13.1 ± 2.1
3.3 In Vitro Assessment of Micelle Stability
3.4 Enhanced Cellular Uptake
3.5 In Vitro Cytotoxicity of dcBSA-147-PEO(5000)28-(DOX)14
4 Summary and Conclusions
The efficient protein-derived drug delivery system dcBSA-147-PEO(5000)28 (2) was prepared based on the polycationic albumin precursor protein BSA-147. This copolymer was designed to encompass several important features such as (1) positive charges to facilitate cellular uptake via endocytosis, (2) a large number of hydrophilic and lipophilic groups along the backbone facilitating the formation of nanosized micelles of about 56 nm with narrow size distributions and high stability in different physiological buffers, cell media and a broad pH range, (3) about 28 PEO chains along the backbone to reduce plasma protein binding, immunogenicity and contribute to high micelle stability in solution, (4) high drug loading of about 14 lipophilic DOX molecules per micelle. Due to the synergistic combination of all these features, unique nanosized containers were achieved that are attractive to efficiently encapsulate and stabilize lipophilic molecules.
Cell imaging via confocal microscopy revealed fast cell uptake of dcBSA-147-PEO(5000)28-(DOX)14 nanocontainers after only 1 h of incubation time. DOX uptake after encapsulation into micelles was fivefold increased compared to free DOX. In vitro cytotoxicity experiments revealed corresponding results since an about five-times higher cytotoxicity of dcBSA-147-PEO(5000)28-(DOX)14 micelles compared to free DOX was found. The exact mechanism of intracellular drug release is still unknown and will be investigated in future studies. The drug delivery system reported herein could in principle be used to encapsulate a broad range of lipophilic drug molecules and might therefore be attractive for combination therapy, e.g. the application of different drugs acting via different modes of action. In addition, the denatured protein backbone provides multiple functional groups available for further chemical modifications. In this way, a highly versatile and attractive platform for anticancer drug delivery was developed with great potential for in vivo studies.
Financial support from NUS start-up grant under Grant No. WBS-R143-000-393-646 and WBS-R143-000-367-133 and the Singapore National Research Foundation proof-of-concept Grant NRF2009-POC001-045 are greatly acknowledged.
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