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Published in final edited form as: J Nanomed Nanotechnol. 2012 Apr 20;Suppl 4:008. doi: 10.4172/2157-7439.S4-008

Optimization of Landscape Phage Fusion Protein-Modified Polymeric PEG-PE Micelles for Improved Breast Cancer Cell Targeting

Tao Wang 1, Valery A Petrenko 2, Vladimir P Torchilin 1,*
PMCID: PMC4594861  NIHMSID: NIHMS702809  PMID: 26451274

Abstract

Amphiphilic landscape phage fusion proteins with high affinity and selectivity towards breast cancer MCF-7 (Michigan Cancer Foundation-7) cells self-assemble with polymeric PEG-PE conjugates to form mixed micelles (phage-micelles) capable of cancer cell-targeted delivery of poorly-soluble drugs. While the PEG corona provides the stability and longevity to the micelles, its presence is a potential steric difficulties for the interaction of phage fusion protein with cell surface targets. We attempted to address this problem by controlling the length of the PEG block and the phage fusion protein quantity, selecting the optimal ones to produce a reasonable retention of the targeting affinity and selectivity of the MCF-7-specific phage fusion protein. Three PEG-PE conjugates with different PEG lengths were used to construct phage- and plain-micelles, followed by FACS analysis of the effect of the PEG length on their binding affinity and selectivity towards target MCF-7 cells using either a MCF-7 cell monoculture or a cell co-culture model composed of target cancer MCF-7 cells and non-target, non-cancer C166 cells expressing GFP (Green Fluorescent Protein). Both, the length of PEG and quantity of phage fusion protein had a profound impact on the targetability of the phage-micelles. Phage-micelles prepared with PEG2k-PE achieved a desirable binding affinity and selectivity. Incorporation of a minimal concentration of phage protein, up to 0.5%, produced maximal targeting efficiency towards MCF-7 cells. Overall, phage-micelles with PEG2k-PE and 0.5% of phage protein represent the optimal formulation for targeting towards breast cancer cells.

Keywords: Drug delivery, Polymeric micelles, PEG-PE, Phage display, Landscape phage fusion protein, Tumor targeting, Breast cancer

Introduction

Lipophilic compounds account for more than 40% of new drug candidate molecules. Despite their potent pharmacological activity, therapeutic application of these molecules is limited due to their poor solubility and low bioavailability. To overcome this limitation, many efforts have been made to develop effective drug delivery systems in order to enhance solubility of hydrophobic drugs [1-4].

One of promising delivery systems is the polymeric micelle. Nano-scale micellar particles can be formed by the self-assembly of amphiphilic molecules in an aqueous environment, with hydrophobic fragments forming the core of a micelle and with the hydrophilic parts forming a micellar corona [5,6]. The hydrophobic core of a micelle has been used to encapsulate a variety of sparingly-soluble therapeutic and diagnostic agents [5-9]. Micellar drug delivery systems substantially increase the bioavailability of poorly-soluble pharmaceuticals and protect them from destructive factors upon parenteral administration [10]. Their nanometre-sizes (typically, between 5 and 50 nm) allow micellar drugs to passively target tumor sites via the Enhanced Permeability and Retention (EPR) effect [11].

The tumor-targeting efficiency of micelle-encapsulated drugs can be further enhanced by introducing targeting ligands into a micellar formulation to allow for active targeting of tumors [6,12]. In the ongoing efforts in the search for targeting ligands, peptide-mediated tumor targeting has become a fast growing field [13], since peptides show multiple advantages, including lesser susceptibility to clearance by the Mononuclear Phagocyte System (MPS), less immunogenicity, and better tumor penetration when compared with antibodies [14,15]. The molecular mechanism behind this phenomenon is that peptide receptors are over-expression in a wide spectrum of tumors [16]. Combinatorial technologies, such as phage display techniques, have made targeting ligands available in a high throughput fashion [17,18].

Recently, we identified MCF-7 breast- and prostate cancer–specific phage fusion proteins and demonstrated their abilities for actively-targeted delivery of the liposomal doxorubicin [19-22]. However, the effectiveness of the use of liposomes for delivery of water-insoluble drugs is far from ideal. Both, the limited drug encapsulation capacity and the potentially unstable packaging of hydrophobic drugs within the lipid bilayers of liposomes are major concerns [5,23]. Additionally, the in vivo premature release of a liposome-loaded hydrophobic drug could represent other issues [23]. We proposed recently the use of PEG-PE-based micelles for the solubilization of hydrophobic drugs [12] and explored the utility of phage fusion proteins in targeting delivery of micelles carrying poorly-soluble drugs. This approach is based on the amphiphilic nature of the phage fusion protein, which ensures its ability to assemble spontaneously with micelle-forming polymers, such as PEG-PE conjugates, resulting in the formation of mixed micelles capable of delivery of water-insoluble drug to specific tumor cells (Figure 1).

Figure 1.

Figure 1

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Schematic representation of the self-assembly of landscape phage fusion protein with polymeric PEG-PE conjugates to form phage-micelles for targeted delivery of hydrophobic drugs.

However, the presence of a PEG corona in the mixed micelles could potentially set up a steric barrier between the targeting phage fusion peptides and molecular receptors on the surface of target cells. In this study, we sought to address this problem by controlling the length of the PEG block and phage protein quantity, selecting parameters, which produce an optimal balance between the target affinity and selectivity of the micelle-incorporated phage protein.

Materials and Methods

Materials and reagents

1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)2000] (ammonium salt; PEG2000-PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)750] (ammonium salt; PEG750-PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)5000] (ammonium salt; PEG5000-PE ), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt, Rho-PE) were purchased from Avanti Polar Lipids Inc (Alabaster, AL). Paclitaxel (PCT) was from Cedarburg Pharmaceuticals (Beverly, MA). Dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific (Pittsburgh, PA). Sodium cholate was from Sigma (St.Louis, MO). Cell Titer Blue assay® kit was from Promega (Madison, WI). Fluor Mounting Medium was from Trevigen Inc (Gaithersburg, MD). MCF-7 human breast adenocarcinoma (HTB-22™) cells and C166-GFP (CRL-2583™) mouse yolk sac endothelial cells were obtained from the ATCC (American Type Culture Collection) (Manassas, VA). All cells were grown as recommended by the ATCC at 37°C, 5% CO2.

Phage fusion protein

Phage selection and phage protein purification have been carried out as described by us previously [19].

Preparation of micellar formulations

Phage-micelles were prepared with compositions shown in Table 1. Briefly, to make rhodamine-labeled micelles, 5 mM of PEG-PE was mixed with traces of rhodamine-PE in chloroform. To form phage-micelles, after evaporation of chloroform, the PEG-PE film formed was hydrated with MCF-7-specific-phage fusion proteins dissolved in 10 mM of sodium cholate, followed by vortexing and overnight dialysis against Phosphate Buffered Saline (PBS), pH 7.4 to remove the detergent. To form plain micelles, the PEG-PE film formed after evaporation was hydrated with PBS, pH 7.4, followed by vortexing and overnight dialysis against PBS, pH 7.4.

Table 1.

Micellar formulations.

The use of micelles Designations PEG length PEG-PE
(mM)		 Phage Protein
(% w/w)
*Optimization of PEG length Phage PEG5K-PE micelles 5K 5 0.5
Phage PEG2K-PE micelles 2K 5 0.5
Phage PEG750-PE micelles 750 5 0.5
Plain PEG5K-PE micelles 5K 5 0
Plain PEG2K-PE micelles 2K 5 0
Plain PEG750-PE micelles 750 5 0
*Optimization of phage protein quantity Plain PEG2K-PE micelles 2K 5 0
Phage PEG2K-PE micelles 2K 5 0.125
Phage PEG2K-PE micelles 2K 5 0.25
Phage PEG2K-PE micelles 2K 5 0.5
Phage PEG2K-PE micelles 2K 5 1
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*

Trace rhodamine-PE was added to formulations

FACS analysis of the uptake of phage-micelles by MCF-7 Cells

MCF-7 cells were grown in 12.5 cm2 flasks in MEM with 10% serum until 70-80% confluence. Cells were incubated with 75 μM of rhodamine-labeled plain- or phage-micelle formulations for 1 h. After washing 3 times with PBS, pH 7.4, cells were detached and collected by centrifugation. The cell pellets were resuspended in 200 μl of PBS with 4% paraformaldehyde, followed by flow cytometry (FACS) analysis. A right shift on the x-axis of the histogram plot indicated the cellular binding of the rhodamine-labeled micelles.

FACS Analysis of Selective Binding of Phage-Micelles with Target MCF-7 Cells in a Co-culture Model

Target MCF-7 cells were co-cultured with non-target C166 endothelial cells expressing GFP in a 1:1 ratio and seeded in 12.5cm2 flasks in MEM with 10% serum. After co-culture until 70-80% confluence, cells were incubated with 75 μM of rhodamine-labeled plain- or phage-micelle formulations for 1 h. The cells were washed 3 times with PBS (pH 7.4), detached, and collected by centrifugation. The cell pellets were resuspended in 200 μl of PBS with 4% paraformaldehyde, followed by the FACS analysis. After acquiring data displayed as dot plots, the dot plots were inserted into four regions (R1, R2, R3, and R4). The cellular binding of the rhodamine-labeled micelles was detected as a right shift of the cell population on the x axis (FL2-H, Red). The percent cell-associated micelles were calculated as follows:

For MCF-7 cells,

The percent cells with associated micelles = R3 / (R1+R3) × 100.

For C166-GFP cells,

The percent of cells with associated micelles = R4 / (R2+R4) × 100.

The binding affinity was determined by the percent of MCF-7 cells with associated micelles.

The binding selectivity was defined as the percent of MCF-7 cells with associated micelles divided by the percent of C166-GFP cells with associated micelles as follows:

The binding selectivity = [R3 / (R1+R3)] / [R4 / (R2+R4)].

Fluorescence microscopy analysis of selective binding of optimized phage-micelles to target MCF-7 cells

MCF-7 and C166-GFP cells were co-cultured on 6-well plates for 24h at 37°C, and treated with 75 μM of rhodamine-labeled phage-micelles in MEM with 10% serum for 30 min at 37°C. After washing 3 times with PBS, the coverslip was mounted onto a glass slide over the fluorescence mounting medium. The images were acquired by a fluorescence microscope (Nikon, Japan) at 40 × magnification with FITC or TRITC filters.

Cytotoxicity

After MCF-7 cells were cultured in 96-well microplates to 50-60% confluence, cells were treated for 72h with paclitaxel (PCT)-loaded, optimized PEG2k-PE phage-micelles and different controls, including free PCT in DMSO, PCT-loaded PEG2k-PE plain micelles, and drug-free, optimized phage-micelles. The concentration of paclitaxel used in different treatments with drug-containing formulations is equivalent to 1.76 μM. Cells were then washed once with PBS, pH 7.4, and incubated with fresh complete medium (100 μl/well) along with the Cell Titer Blue assay reagent (20 μl/well) for 2 h at 37°C. The fluorescence intensity was measured using a multi-detection microplate reader (Bio-Tek, Winooski, VT) with 525/590 nm excitation/emission wavelengths.

Results

Optimization of the PEG Length

We have used three PEG species with different lengths to construct plain- and phage-micelles, and compared their binding affinity towards target MCF-7 cells. As expected, a decrease in the length of PEG chain increased target cell association of phage-micelles in the order of PEG750 > PEG2k > PEG5k (Figure 2A). Control plain micelles with varying PEG chain also showed their binding affinity to MCF-7 cells in the order of P EG750 > PEG2k> =PEG5k (Figure 2B). However, the modification of PEG5k micelles (Figure 2C) and PEG750 (Figure 2E) micelles with MCF-7 specific phage fusion protein showed less advantage in tumor-cell targeting compared to non-modified plain micelles. In contrast, the phage protein-modified PEG2k-PE micelles (Figure 2D) showed a significantly enhanced target cell binding compared to plain PEG2k-PE micelles.

Figure 2. FACS analysis of the effect of different PEG length on the binding affinity of micellar formulations towards target MCF-7 cells.

Figure 2

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MCF-7 breast cancer cells were treated for 1h with either plain micelles or phage-micelles constructed from PEG750-PE, PEG2k -PE, or PEG5k -PE conjugates, followed by FACS analysis of cell-associated micelles as indicated by an increase in red fluorescence intensity (FL2-H).

To investigate the binding selectivity of different micelle formulations towards target MCF-7 cancer cells compared to non-target, non-cancer cells, we have designed a co-culture assay, in which target cancer MCF-7 cells were co-grown with non-target, non-cancer endothelial cells, C166 cells expressing GFP (C166-GFP). FACS analysis of the co-culture revealed two distinct cell populations. One cell population corresponding to higher green fluorescence intensity, as indicated on the Y-axis (FL1-H) of the dot plot, was C166-GFP cells. The other cellular population with lower green fluorescence intensity was MCF-7 cells (Figure 3A). After Treatment of this co-culture with different micelles followed by the FACS analysis, we found that plain- and phage- PEG750-PE micelles had the highest binding to target cells but also to non-target cells, indicating a poor selective binding to tumor cells (Figure 3B and Figure 3E), while micelles with PEG5K-PE showed the best targeting selectivity but a compromised binding affinity (Figure 3D and Figure 3G). Micelles with PEG2k-PE had an optimal balance between the binding affinity and selectivity (Figure 3C, Figure 3F and Figure 4).

Figure 3. FACS analysis of cellular binding of micellar formulations prepared with different PEG blocks in a co-culture system.

Figure 3

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A co-culture composed of target MCF-7 cells and non-target C166-GFP cells was treated for 1h with either plain micelles or phage-micelles constructed from PEG750-PE, PEG2k-PE, or PEG5k-PE conjugates, followed by FACS analysis of cell-associated micelles as indicated by the red fluorescence increase. The dot plots were bounded into four regions (R1, R2, R3, and R4). FL1-H (green); FL2-H (red); (A) A representative dot plot showing the untreated co-culture of MCF-7 cells and C166-GFP cells. Red dots in region R1, the location of untreated MCF-7 cells; green dots in region R2, the location of untreated C166-GFP cells; (B-D) Plain micelle-treated co-culture of MCF-7 cells and C166-GFP cells. Pink dots in region R3, plain-micelle-associated MCF-7 cells; blue dots in region R4, plain-micelle-associated C166-GFP cells; (E-G) MCF-7-targeted phage-micelle-treated co-culture of MCF-7 and C166-GFP cells. Pink dots in region R3, phage-micelle-associated MCF-7 cells; blue dots in R4, phage-micelle-associated C166-GFP cells.

Figure 4. Quantitative analysis of the effect of PEG length on the binding affinity and selectivity of micellar formulations after FACS analysis in a co-cultured cell model.

Figure 4

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(A) The percent of cells with associated micelles indicated as the cell population shifted to the region with higher red fluorescence intensity in the dot plot acquired by FACS analysis. (B) The binding affinity defined as the percent of MCF-7 cells with associated micelles divided by the total MCF-7 cells tested; The binding selectivity defined as the percent of MCF-7 cells with associated micelles divided by the percent of C166-GFP cells with associated micelles (mean ± SD, n=9).

Optimization of phage protein quantity

We defined an optimal phage protein quantity as one providing the maximal binding affinity with a minimum of phage fusion protein used. The incorporation of MCF-7-targeting phage fusion protein up to 0.25% by weight into PEG2k-PE micelles resulted in a limited increase in target cell-association compared to the plain PEG2k-PE micelles, but the modification of plain micelles with 0.5% phage fusion protein provided a pronounce enhancement in target MCF-7 cell-binding, but the further increase in phage protein quantity from 0.5% to 1% by weight did not result in any noticeable increase in the binding (Figure 5). Therefore, the optimal phage protein quantity to be used in phage-micelles is 0.5% by weight.

Figure 5.

Figure 5

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Effect of phage protein quantity on the binding affinity of micelle formulations towards target MCF-7 cells (Mean ± SD, n=6).

Targetability and cytotoxicity of the optimized phage-micelle formulation

After treatment of the co-culture composed of target MCF-7 and non-target C166-GFP with rhodamine-labeled phage-micelles, the overlay fluorescence micrograph clearly showed the lack of co-localization between red and green fluorescence, indicating that the optimized phage-micelles preferentially bind to target MCF-7 cancer cells rather than to co-cultured C166-GFP cells (Figure 6A).

Figure 6. Targetability and cytotoxicity of the optimized phage-micelle formulation.

Figure 6

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(A) Selective cell binding by the florescence microscopy. Bright field shows a co-culture of MCF-7 and C166-GFP cells. In the overlay image, green fluorescence shows the location of C166-GFP cells; red fluorescence shows rhodamine-labeled phage-micelles associated with cells. (B) Cytotoxicity of different formulations towards MCF-7 cells after 72 h treatment with MCF-7-targeted phage-micelles and various controls, including free PCT in DMSO, PCT-loaded plain micelles, drug-free MCF-7-targeted phage-micelles. (* p<0.05, mean ± SD, n=6).

The treatment with the optimized phage-micelles loaded with paclitaxel at a PCT concentration of 1.76 μM for 72 h, led to 51.3 % MCF-7 cell death. The estimated IC50 of the phage micelle-loaded with paclitaxel is 1.63 μM (as PCT). The significantly lower tumor cell killing is seen however in the groups treated with both, free paclitaxel dissolved in DMSO and paclitaxel in plain PEG2k-PE micelles at the equivalent concentration of PCT tested, with MCF-7 cell death level at 18.7% and 23.8%, respectively (Figure 6B). The drug-free, optimized phage-micelles produced negligible tumor cell killing.

Discussion

Combining of passive tumor targeting of pharmaceutical nanocarriers with tumor cell recognition systems represents a sophisticated strategy for targeted delivery of anti-cancer drugs to specific tumor cells [24]. Within this approach, a novel self-assembled micellar system, composed of PEG-diacyllipid micelles and phage-derived fusion proteins, has recently been designed for tumor-targeted delivery of water-insoluble drugs, such as paclitaxel [12].

The PEG-diacyllipid micelles prepared from amphiphilic polymer conjugates of polyethylene glycol (PEG) and diacyllipids (PE) have served as drug carriers for the solubilization of water-insoluble drugs and for tumor passive targeting [5,6,10]. Compared with conventional amphiphilic polymer micelles, the use of diacyllipid moieties (PE) as hydrophobic blocks, offers better particle stability as a result of a considerable contribution of the two fatty acid acyls within the PE blocks that increase hydrophobic interactions within the micelle’s core. On the other hand, highly water-soluble PEG chains effectively provide steric protection and physiological stability for various nanoparticles in biological media. PEG chains protect nanoparticles from the clearance by the MPS system, and from other possible undesirable interaction with blood components. In the blood stream, PEGylated particles have a longer circulation time, which additionally promotes drug accumulation within target sites, such as tumors, via the EPR effect. Consequently, PEG-PE micelles loaded with a variety of poorly-soluble drugs can deliver their payload into tumors in mice with a greater efficiency [25,26].

It has been recognized for some time that PEGylation could also influence drug delivery negatively [27,28]. The presence of the PEG corona covering drug carriers produces a steric hindrance, affecting the interaction of drug carriers with target cells [28]. Particularly, PEG blocks within the micelles modified with phage fusion protein could have interfered with the interaction of targeting phage fusion protein with tumor cell-surface receptors.

An important method to solve this PEG dilemma is to control PEG length to induce a proper balance between the micellar stability and its targetability. Early studies have shown the effect of PEG length on micellar size and thermodynamic stability [5]. Generally, PEG-PE conjugates with PEG blocks with a molecular weight from 1000 to 15000 Dalton are able to form stable nanoparticles. With an increase in the length of PEG form 750 to 2000 to 5000 Dalton, particle sizes are increased and the Critical Micellar Concentration (CMC) of PEG-PE micelles is decreased [5]. Further studies have demonstrated that PEG length also dictates the in vivo behaviour of PEG-PE micelles. Micelles formed with PEG5K-PE have a longer circulation time, with less uptake by normal tissue compared to micelles prepared from a shorter PEG-PE conjugate, and a higher accumulation in tumors compared to non-target tissues, such as muscle, as observed in a Lewis lung carcinoma-bearing experimental mice [29,30]. Our results have further demonstrated the shielding effect produced by the PEG chain. The longer the PEG block used, the weaker the interaction of PEG-PE micelles with tumor cells. On the other hand, micelles prepared from PEG-PE conjugates with longer versions of PEG have better targeting selectivity towards tumor cells compared to non-target, non-cancer cells.

Within the objective of this study, to develop an optimized self-assembling system for active tumor targeting, we used the whole landscape phage fusion protein (the targeting peptide fused to phage major coat protein PVIII), the hydrophobic phage major coat protein part of which interacts with diacyllipid-PE to form the micellar core. Targeting peptides of the whole protein, together with PEG block of PEG-PE conjugates, built up the micellar corona. An ideal phage-micelle formulation should contain an appropriate PEG block, which can efficiently shield hydrophobic segments (PE and phage major coat protein PVIII), and, at the same time, exposes the targeting peptides to a maximally possible extent. During optimization of the PEG length and phage protein quantity used to formulate the targeted phage-micelles, we found that only PEG2K-PE increased binding affinity of phage-micelles towards targeted cells, implying that PEG750 was too short to hide the hydrophobic segments, while PEG5K was too long to allow for the exposure of targeting ligands. In accordance with this observation, PE G750-PE micelles showed pronounced non-specific binding to non-target C166-GFP cells. Interestingly, the increase in phage protein quantity in PEG2K-PE phage-micelles was not necessarily favourable for targeting, suggesting that the mixed micellar aggregations can only accommodate a certain amount of phage fusion protein within stable particles with a maximal tumor cell targeting. Similar phenomenon was also observed when the phage protein was used to modify liposomes [31].

Loading hydrophobic drug paclitaxel into the optimized formulation with PEG2K-PE and 0.5% of phage fusion protein (by weight) forms stable nanoparticles. Incorporation of phage protein into PEG2K-PE micelles improves MCF-7 tumor cell targeting and cytotoxicity of the micellar paclitaxel [12]. Similarly, we have earlier observed the specific tumor cell targeting and enhanced tumor cell killing when liposomes are modified with phage fusion protein for the delivery of water-soluble amphiphilic drugs, such as doxorubicin hydrochloride [19].

The PEG2K-DSPE used in the preparation of the optimized phage-micelles has been approved as an excipient by the U.S. Food and Drug Administration, and it is generally safe, biocompatible and relatively nontoxic [32,33]. Thorough investigation on the metabolism and excretion of PEG and PEG-biological molecule conjugates suggests the less safety concerns associated with their utility in chronic and acute administration even with high concentration [34]. In this study, drug-free PEG-PE micelles modified with phage fusion protein showed no toxicity towards MCF-7 at the concentration of PEG-PE up to 48 μM, providing evidence for the safe use of the phage-micelle formulation.

In summary, phage-micelles created by the self-assembly of phage fusion protein and PEG-PE specifically bind to targeted tumor cells. Both length of PEG and quantity of phage fusion protein have a profound impact on the micellar targetability. PEG2K-PE and phage protein quantity of 0.5% (w/w) constitute a formulation with an optimized balance between tumor cell-binding affinity and selectivity.

Acknowledgements

This work was supported by NIH grant # R01 CA125063-01 and the Animal Health and Disease Research grant 2006-9, College of Veterinary Medicine, Auburn University to Valery A. Petrenko and by NIH grants U54CA151881 to Vladimir P. Torchilin.

References

  • 1.Merisko-Liversidge EM, Liversidge GG. Drug nanoparticles: formulating poorly water-soluble compounds. Toxicol Pathol. 2008;36:43–48. doi: 10.1177/0192623307310946. [DOI] [PubMed] [Google Scholar]
  • 2.Dahan A, Hoffman A. Rationalizing the selection of oral lipid based drug delivery systems by an in vitro dynamic lipolysis model for improved oral bioavailability of poorly water soluble drugs. J Control Release. 2008;129:1–10. doi: 10.1016/j.jconrel.2008.03.021. [DOI] [PubMed] [Google Scholar]
  • 3.Brewster ME, Loftsson T. Cyclodextrins as pharmaceutical solubilizers. Adv Drug Deliv Rev. 2007;59:645–666. doi: 10.1016/j.addr.2007.05.012. [DOI] [PubMed] [Google Scholar]
  • 4.Porter CJ, Trevaskis NL, Charman WN. Lipids and lipid-based formulations: optimizing the oral delivery of lipophilic drugs. Nat Rev Drug Discov. 2007;6:231–248. doi: 10.1038/nrd2197. [DOI] [PubMed] [Google Scholar]
  • 5.Torchilin VP. Micellar nanocarriers: pharmaceutical perspectives. Pharm. 2007;24:1–16. doi: 10.1007/s11095-006-9132-0. [DOI] [PubMed] [Google Scholar]
  • 6.Torchilin VP. Targeted pharmaceutical nanocarriers for cancer therapy and imaging. AAPS J. 2007;9:128–147. doi: 10.1208/aapsj0902015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Danson S, Ferry D, Alakhov V, Margison J, Kerr D, et al. Phase I dose escalation and pharmacokinetic study of pluronic polymer-bound doxorubicin (SP1049C) in patients with advanced cancer. Br J Cancer. 2004;90:2085–2091. doi: 10.1038/sj.bjc.6601856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Matsumura Y, Hamaguchi T, Ura T, Muro K, Yamada Y, et al. Phase I clinical trial and pharmacokinetic evaluation of NK911, a micelle-encapsulated doxorubicin. Br J Cancer. 2004;91:1775–1781. doi: 10.1038/sj.bjc.6602204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Kim TY, Kim DW, Chung JY, Shin SG, Kim SC, et al. Phase I and pharmacokinetic study of Genexol-PM, a cremophor-free, polymeric micelle-formulated paclitaxel, in patients with advanced malignancies. Clin Cancer Res. 2004;10:3708–3716. doi: 10.1158/1078-0432.CCR-03-0655. [DOI] [PubMed] [Google Scholar]
  • 10.Sutton D, Nasongkla N, Blanco E, Gao J. Functionalized micellar systems for cancer targeted drug delivery. Pharm Res. 2007;24:1029–1046. doi: 10.1007/s11095-006-9223-y. [DOI] [PubMed] [Google Scholar]
  • 11.Lammers T, Hennink WE, Storm G. Tumour-targeted nanomedicines: principles and practice. Br J Cancer. 2008;99:392–397. doi: 10.1038/sj.bjc.6604483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Wang T, Petrenko VA, Torchilin VP. Paclitaxel-loaded polymeric micelles modified with MCF-7 cell-specific phage protein: enhanced binding to target cancer cells and increased cytotoxicity. Mol Pharm. 2010;7:1007–1014. doi: 10.1021/mp1001125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Reubi JC. Peptide receptors as molecular targets for cancer diagnosis and therapy. Endocr Rev. 2003;24:389–427. doi: 10.1210/er.2002-0007. [DOI] [PubMed] [Google Scholar]
  • 14.Mori T. Cancer-specific ligands identified from screening of peptide-display libraries. Curr Pharm Des. 2004;10:2335–2343. doi: 10.2174/1381612043383944. [DOI] [PubMed] [Google Scholar]
  • 15.Thurber GM, Schmidt MM, Wittrup KD. Factors determining antibody distribution in tumors. Trends Pharmacol Sci. 2008;29:57–61. doi: 10.1016/j.tips.2007.11.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Joshiand BP, Wang TD. Exogenous Molecular Probes for Targeted Imaging in Cancer: Focus on Multi-modal Imaging. Cancers (Basel) 2010;2:1251–1287. doi: 10.3390/cancers2021251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Petrenko V. Evolution of phage display: from bioactive peptides to bioselective nanomaterials. Expert Opin Drug Deliv. 2008;5:825–836. doi: 10.1517/17425247.5.8.825. [DOI] [PubMed] [Google Scholar]
  • 18.Landon LA, Deutscher SL. Combinatorial discovery of tumor targeting peptides using phage isplay. J Cell Biochem. 2003;90:509–517. doi: 10.1002/jcb.10634. [DOI] [PubMed] [Google Scholar]
  • 19.Wang T, D’Souza GG, Bedi D, Fagbohun OA, Potturi LP, et al. Enhanced binding and killing of target tumor cells by drug-loaded liposomes modified with tumor-specific phage fusion coat protein. Nanomedicine (Lond) 2010;5:563–574. doi: 10.2217/nnm.10.30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Wang T, Kulkarni N, D’Souza GG, Petrenko VA, Torchilin VP. On the mechanism of targeting of phage fusion protein-modified nanocarriers: only the binding peptide sequence matters. Mol Pharm. 2011;8:1720–1728. doi: 10.1021/mp200080h. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Jayanna PK, Bedi D, Gillespie JW, DeInnocentes P, Wang T, et al. Landscape phage fusion protein-mediated targeting of nanomedicines enhances their prostate tumor cell association and cytotoxic efficiency. Nanomedicine. 2010;6:538–546. doi: 10.1016/j.nano.2010.01.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Wang T, Yang S, Petrenko VA, Torchilin VP. Cytoplasmic delivery of liposomes into MCF-7 breast cancer cells mediated by cell-specific phage fusion coat protein. Mol Pharm. 2010;7:1149–1158. doi: 10.1021/mp1000229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Drummond DC, Meyer O, Hong K, Kirpotin DB, Papahadjopoulos D. Optimizing liposomes for delivery of chemotherapeutic agents to solid tumors. Pharmacol Rev. 1999;51:691–743. [PubMed] [Google Scholar]
  • 24.Noble CO, Kirpotin DB, Hayes ME, Mamot C, Hong K, et al. Development of ligand-targeted liposomes for cancer therapy. Expert Opin Ther Targets. 2004;8:335–353. doi: 10.1517/14728222.8.4.335. [DOI] [PubMed] [Google Scholar]
  • 25.Constantinides PP, Chaubal MV, Shorr R. Advances in lipid nanodispersions for parenteral drug delivery and targeting. Adv Drug Deliv Rev. 2008;60:757–767. doi: 10.1016/j.addr.2007.10.013. [DOI] [PubMed] [Google Scholar]
  • 26.Lukyanov AN, Torchilin VP. Micelles from lipid derivatives of water-soluble polymers as delivery systems for poorly soluble drugs. Adv Drug Deliv Rev. 2004;56:1273–1289. doi: 10.1016/j.addr.2003.12.004. [DOI] [PubMed] [Google Scholar]
  • 27.Wang T, Upponi R, Torchilin VP. Design of multifunctional non-viral gene vectors to overcome physiological barriers: Dilemmas and strategies. Int J Pharm. 2012 doi: 10.1016/j.ijpharm.2011.07.013. [DOI] [PubMed] [Google Scholar]
  • 28.Hatakeyama H, Akita H, Harashima H. A multifunctional envelope type nano device (MEND) for gene delivery to tumours based on the EPR effect: a strategy for overcoming the PEG dilemma. Adv Drug Deliv Rev. 2011;63:152–160. doi: 10.1016/j.addr.2010.09.001. [DOI] [PubMed] [Google Scholar]
  • 29.Weissig V, Whiteman KR, Torchilin VP. Accumulation of protein-loaded long-circulating micelles and liposomes in subcutaneous Lewis lung carcinoma in mice. Pharm Res. 1998;15:1552–1556. doi: 10.1023/a:1011951016118. [DOI] [PubMed] [Google Scholar]
  • 30.Torchilin VP. Targeted polymeric micelles for delivery of poorly soluble drugs. Cell Mol Life Sci. 2004;61:2549–2559. doi: 10.1007/s00018-004-4153-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Wang T, Kulkarni N, Bedi D, D’Souza GG, Papahadjopoulos-Sternberg B, et al. In vitro optimization of liposomal nanocarriers prepared from breast tumor cell specific phage fusion protein. J Drug Target. 2011;19:597–605. doi: 10.3109/1061186X.2010.550920. [DOI] [PubMed] [Google Scholar]
  • 32.Krishnadas A, Rubinstein I, Onyuksel H. Sterically stabilized phospholipid mixed micelles: in vitro evaluation as a novel carrier for water-insoluble drugs. Pharm Res. 2003;20:297–302. doi: 10.1023/a:1022243709003. [DOI] [PubMed] [Google Scholar]
  • 33.Onyuksel H, Ikezaki H, Patel M, Gao XP, Rubinstein I. A novel formulation of VIP in sterically stabilized micelles amplifies vasodilation in vivo. Pharm Res. 1999;16:155–160. doi: 10.1023/a:1018847501985. [DOI] [PubMed] [Google Scholar]
  • 34.Webster R, Didier E, Harris P, Siegel N, Stadler J, et al. PEGylated proteins: evaluation of their safety in the absence of definitive metabolism studies. Drug Metab Dispos. 2007;35:9–16. doi: 10.1124/dmd.106.012419. [DOI] [PubMed] [Google Scholar]

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