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Madhu Smitha Harihara Iyer 1 Nanoparticles in Targeted Drug Delivery for Cancer Therapy: Folate Receptor Endocytosis Madhu Smitha Harihara Iyer [email protected] BioE 494 – Atomic & Molecular Nanotechnology BioE 494 – Atomic and Molecular Nanotechnology Fall 2007 Madhu Smitha Harihara Iyer 2 Abstract Treatment of cancer involves using specific chemical agents or drugs called chemotherapeutics to selectively destroy malignant cells or tissues. The targetability of the treatment determines the improvement in the patient’s quality of life and eventual life expectancy. The selectivity in current treatment is predominantly based on the inherent nature of the chemotherapeutic drugs to work on a particular type of cancer cell more intensely than on healthy cells. An effective way of targeting delivery of drugs to cancer cells is by conjugation of folic acid moiety to stable carrier particles which can encapsulates the therapeutic drug. The potential use of nano-sized particles as drug carriers is because of their small size and uniform biodistribution profile. Table of Content Abstract ....................................................................................................................... 2 Table of Content ......................................................................................................... 2 1. Introduction ............................................................................................................. 2 1.1. Nanotechnology in Cancer Therapy .................................................................... 3 2. Nano-Sized Drug Carriers....................................................................................... 4 2.1. Carbon Nanotubes ................................................................................................ 4 2.2. Dendrimers ........................................................................................................... 5 2.3. Gadolinium Nanoparticles ................................................................................... 5 2.4. Gold Nanoparticles .............................................................................................. 6 2.5. Liposomes ............................................................................................................ 7 2.6. Nano Polymers ..................................................................................................... 7 2.7. Protein Cages ....................................................................................................... 8 3. Characterization of Nanoparticles........................................................................... 8 3.1. Dynamic Light Scattering (DLS) ......................................................................... 8 3.2. Sedimentation Equilibrium (SE) .......................................................................... 9 3.3.Sedimentation Velocity (SV) ................................................................................ 9 3.4. Zeta Potential (ζ ) ............................................................................................... 10 3.5. Transmission Electron Microscopy (TEM) ....................................................... 11 3.6. Atomic Force Microscopy (AFM) ..................................................................... 11 4. Cell internalization of Nano-carriers .................................................................... 11 5. MRI Compatible Nanoparticles ............................................................................ 13 References ................................................................................................................. 16 1. Introduction Treatment of cancer involves using specific chemical agents or drugs called chemotherapeutics to selectively destroy malignant cells or tissues. Current cancer therapy consists of three stages of treatment namely the initial chemotherapy to shrink any cancer present, removal of tumor by surgery, and finally more chemotherapy and radiation to fully eradicate the cancer cells [1]. The BioE 494 – Atomic and Molecular Nanotechnology Fall 2007 Madhu Smitha Harihara Iyer 3 effectiveness of the treatment is directly related to the treatment’s ability to target and kill the cancer cells while affecting fewer healthy. The targetability of the treatment determines the improvement in the patient’s quality of life and eventual life expectancy. The selectivity in current treatment is predominantly based on the inherent nature of the chemotherapeutic drugs to work on a particular type of cancer cell more intensely than on healthy cells. Administering bolus doses of the drugs initiate intense side effects dismaying the patients from continuing therapy. An effective way of delivering drugs to cancer cells was facilitated by discovery of the pathway of vitamin uptake in plant cells [2]. This method of cellular uptake called Receptor Mediated Endocytosis (RME) was further studied to deliver macromolecules into animal cells. The study of RME in human pharyngeal cancer cells (KB cells) [3] lead to the discovery of upregulation of folate receptors in cancer cells compared to normal cells. This paved way for a break through in the cancer therapy research. Efforts were now oriented towards possible conjugation of folic acid to antineoplastic drugs and targeting their delivery to cancer cells. But studies revealed that direct conjugation of folic acid to bioactive molecules reduced anticancer activity of the drug and/or alteration of the function of the conjugate. Conjugation of the folate moiety to stable carrier particles which can encapsulates the therapeutic drug provided a feasible means to overcome this problem of ligand conjugation. Hence the use of drug delivery system was introduced to cancer therapy. Conjugating targeting moieties to the drug delivery vehicles increased the likelihood of the drug reaching the tumoral site and minimized the uptake by non-target cells. Other advantages of using drug delivery systems are (i) the drug remains concentrated in the carriers instead of diffusing throughout the body (ii) lower doses can be administered which alleviates side effects and reduction in cost of treatment and (iii) slow release of the drug in the target site. Direct delivery of the drug to the cancer site and prolonged exposure to the drug increased the effectiveness of the therapy. 1.1. Nanotechnology in Cancer Therapy With the advent of Nanotechnology, research in cancer therapy took on new dimensions. The use of nano-sized particles for cancer therapy became an area of intense research. This was because of the potential advantages of using nano-sized particles as drug delivery systems. The extremely small size (<5.0 nm diameter) of these nanoparticles allow easy escape of the vasculature to target tumor cells and metabolic clearance [4]. Current research in cancer therapy is expanding the domain of nano-sized drug delivery systems by experimenting potential nanoparticles for targeted treatment. This paper is a summary of these potential nano-sized drug delivery systems for cancer treatment, their production, characterization and in vitro tumoral activity measurement. BioE 494 – Atomic and Molecular Nanotechnology Fall 2007 Madhu Smitha Harihara Iyer 4 2. Nano-Sized Drug Carriers The use of nanoparticles in cancer therapy is identified by the feasibility of its conjugation to folic acid ligands. This can be assessed by the method of production and characterization of the nanoparticles. Some of the commonly researched nanoparticles for cancer-drug delivery are shown in Figure 1. Figure 1. Nano-sized drug carriers used in cancer research 2.1. Carbon Nanotubes Selective cancer cell destruction can be achieved by functionalization of Single Walled Carbon Nanotubes (SWNTS) with a folate moiety. This enables selective internalization by folate receptor tumor markers. The unique property of SWNTs to absorb light at Near-Infra Red (NIR) wavelength can be used to destroy cancerous cells without harming normal tissue since biological tissues are transparent to light at the NIR wavelength (700-1000 nm). Folic acid is conjugated to the surface of the SWNTs using phospholipids of poly(ethylene glycol) (PL-PEG-FA). To facilitate fluroscent spectroscopic imaging Fluorescein isothiocynate (FITC) moieties can also conjugated in similar fashion. A 1:1 ratio mixture of PL-PEG-FA and PL-PEGFITC is used to produce funtionalized SWNTs using sonication and centrifugation. The small bundles of PL-PEG functionalized SWNTs are characterized by UV-visible-NIR spectroscopy and Atomic Force Microscopy [5]. BioE 494 – Atomic and Molecular Nanotechnology Fall 2007 Madhu Smitha Harihara Iyer 5 Figure 2. Chemical structure of PL-PEG-FA and PLPEG-FITC synthesized by conjugating PL-PEG-NH2 The stability of the non-covalent absorption of PL-PEG molecules onto the sidewalls of SWNTs is studied by analyzing the aggregation of precipitation of the nanoparticles in water and physiological buffers at room temperature and above the physiological temperature (>80°C). 2.2. Dendrimers Generation 5 Polyamidoamine (PAMAM) dendrimers are stable, non-immunogenic and contain ample reactive sites (110 to 128 primary amines) on the surface for conjugation of multiple chemical moieties like targeting ligands, radiopharmaceuticals, dyes and contrast agents [4]. These primary amine groups are known to bond poorly with folic acid due to localized aggregation of the dendrimer branches. But when capped with acetamide functional group these nanoparticles can be effectively conjugated to FA moiety and also bond to the folate receptors in the cancer cells. The study of modification of the functional group in the dendrimer can be done in situ using Molecular Dynamic Simulations. The conformational structure of the generation 5 PAMAM, fluorescein, folic acid nano-sized drug carrier (G5-FITC-FA) can be modeled in the hydrated state to find modifications of the primary amine groups that can optimize targeting [4]. 2.3. Gadolinium Nanoparticles Neutron Capture Therapy (NCT) is a potential cancer therapy which utilizes a stable nuclide delivered to tumor cells that can produce localized cytotoxic radiations when irradiated by thermal or epithermal neurons [7]. Gadolinium (Gd) is a prospective NCT agent which can be effectively used for treating cancer because of (i) its large neutron capture cross section area which requires only shorter neutron irradiation times and (ii) Gd-neutron capture reaction leading to emission of photons with tumor killing energy deposition at longer ranges in tissues and (iii) its additional use as contrast agent in magnetic resonance imaging (MRI). Nano-sized Gd particles can be synthesized from oil-in-water microemulsion templates and conjugated to PEG attached folate moieties [7]. The size characterization of the Gd-PEG-FA nanoparticles can be done using Transmission Electron Microscopy as shown in Figure 3. BioE 494 – Atomic and Molecular Nanotechnology Fall 2007 Madhu Smitha Harihara Iyer 6 Figure 3. Transmission Electron Microscopy of Gd-PEG-FA nanoparticles 2.4. Gold Nanoparticles Gold nanoparticles (AuNP) soluble over a broad range of pH and ionic strength values can be prepared by nanoprecipitation using centrifugation technique [10]. The conjugation of FA to AuNP is achieved indirectly through bonding with thiotic acid and Poly(ethylene glycol) (PEG) polymer chains. The use of different PEG backbones can address the method of noncovalant bonding between the AuNPs and the reactive functional groups present in the PEG spacers [9]. Figure 4 shows the PEG backbones that can bind to AuNPs through different mode of bonding namely (i) electrostatic attraction of negatively charged AuNPs with positively charged PEG molecules, (ii) hydrophobic interaction between PEGs and AuNPs, and (iii) covalent bonding between the thiols/amine groups present in different PEGs and the AuNPs. Figure 4. Chemical Structure of Different PEG backbones BioE 494 – Atomic and Molecular Nanotechnology Fall 2007 Madhu Smitha Harihara Iyer 7 2.5. Liposomes Lipsomes are most commonly used nanoparticles that are conjugated with antibodies/proteins to specifically target cancer cells [12]. The use of folic aicd ligand for targeting cancer cells has many advantages over the traditional use of antibodies (i) folate is non allergenic while antibodies may elicit an immune response (ii) folate mediates liposome endocytosis into non-lysosomal compartments whereas antibodies frequently promote uptake into lysosomes (iii) folate is compatible with PEG-derivatized lipids in contradistinction to antibody conjugated liposomes, which lose their binding affinity upon incorporation of PEG lipids and (iv) folate ligand is inexpensive, stable during storage and in vivo circulation, intrinsically nontoxic to cells, and easy to conjugate to the desired liposomes [13]. Unmodified liposomes, when injected into the blood stream do not survive long in circulation because of their nonspecific recognition and removal by macrophages of the reticuloendothelial system. PEG coating is believed to inhibit this nonspecific adsorption and hence survivability of liposomes can be improved by conjugating PEG molecules to their surface. But these PEG spacer molecules are longer than folate moiety and thus reduce the effective targetability by occlusion [11]. But attaching the folic acid ligands to the distil end of the lipid conjugated PEG molecules enhances flexibility and targetability of the folic acid since they are free to extend away from the liposome surface to probe different regions of a cell surface. 2.6. Nano Polymers Nano polymers are prepared by breaking down of macro-scale copolymers by sonification or formation of emulsion templates followed by nanoprecitipation and centrifugation [14]. Some nanopolymers are formed by self-emulsification of the polymer chain dissolved in a solute by using emulsifiers. Poly(lactide-co-glycolide) [PLGA] nanopolymers are synthesized by selfemulsification from Dichloromethane solute due to the presnece of vitamin E or TPGS [αtocopheryl succinate esterified to polyethylene glycol] moiety [15]. Surface hydrophilicity is an important factor to be considered for increasing the blood circulation time of the carrier of the nanoparticles [17]. Hence the nanopolymers are conjugated to PEG moiety to reduces opsonization of after intravenous administration. The Shell Cross-Linked (SCK) copolymers have a unique amphiphilic core-shell morphology. They are characterized by their structural integrity and functionality to attach receptor specific ligands on the shell surface [20]. BioE 494 – Atomic and Molecular Nanotechnology Fall 2007 Madhu Smitha Harihara Iyer 8 2.7. Protein Cages Hibiscus chlorotic ringspot virus (HCRSV) is monodispersed to produce nanosized protein cages which can be used to encapsulate doxorubicin, an anticancer drug, and target cancer cells by conjugation with folic acid [19]. Quantitative characterization of the protein cage nano-carrier can be calculated using the following equations Equation 1: Loading efficiency (%) = Weight doxorubicin loaded/Weight protein cage Equation 2: Encapsulation efficiency (%) = Weight Equation 3: Reassembly efficiency (%) = Weight doxorubicin loaded X 100 /Weight total doxorubicin used X 100 protein cage/Weight total coat protein used X 100 Equation 4: Number of doxorubicin molecules encapsidated within each capsid (N), N = LE/Mw doxorubicin X (Mw coat protein X 180) where Mw doxorubicin and Mw coat protein values were 545 and 37,000 respectively. 3. Characterization of Nanoparticles The size, distribution, stability and folic acid conjugation potential of the nanoparticles are determined by various characterization techniques. Different characterization techniques are used to deteremine different properties of the nanopartcles. 3.1. Dynamic Light Scattering (DLS) The hydrodynamic diameter distribution of the nanoparticles and their distribution averages in a solution are determined using dynamic light scattering. Nanoparticles aree dialyzed into 50 mM Lead Sulphite solution at pH 7.1 prior to analysis. The buffered nanoparticle solutions are either centrifuged or filtered to remove dust particles. Incident laser light is set to obtain photon counting rate between 200 & 300 kcps and the scattered light is collected at a fixed angle of 90°. The calculations of the nanoparticle diameter distributions and distribution averages are doen using single-exponential fitting, cumulants analysis, and non-negatively constrained least-squares particle size distribution analysis routines. The diffusion coefficient, d, can be corrected to standard conditions, d20,w, using the following equation: Equation 5: d20,w = d* ηb/ ηw where, ηw is the absolute viscosity of water at 20 °C (1.002 cp) and ηb is the absolute viscosity of the buffer at 20°C calculated from viscosity increments [20]. BioE 494 – Atomic and Molecular Nanotechnology Fall 2007 Madhu Smitha Harihara Iyer 9 3.2. Sedimentation Equilibrium (SE) Sedimentation equilibrium is an analytical ultracentrifugation method for measuring protein molecular masses in solution and for studying protein-protein interactions. Particular applications of this technique are (i) establishing the native state of a protein as a monomer, dimer, trimer, etc (ii) measuring the equilibrium constant for association of proteins which reversibly self-associate to form oligomers (iii) measuring the stoichiometry of complexes between two or more different proteins (e.g. a soluble receptor and its ligand or an antigen-antibody pair), or between a protein and a non-protein ligand (iv) measuring the equilibrium constants for reversible protein-protein and protein-ligand interactions (approximate range 1 nanomolar to 1 millimolar). The partial specific volume (n) for the nanoparticles can be determined via sedimentation equilibrium analysis for protonated and deuterated buffer solutions of the nanoparticles. 3.3.Sedimentation Velocity (SV) Sedimentation velocity is an analytical ultracentrifugation (AUC) method that measures the rate at which molecules move in response to centrifugal force generated in a centrifuge. This sedimentation rate provides information for (i) mass and conformational homogeneity of the sample (ii) aggregation in protein samples and quantifying the amount of aggregate (iii) comparing conformations for samples from different lots, manufacturing processes, or expression systems (iv) determining the overall shape of non-glycosylated protein molecules in solution (v) measuring the distribution of sizes in samples (vi) detecting changes in protein conformation and (vii) studying the formation and stoichiometry of tight complexes between proteins. The sedimentation time-derivative determined ƒc/ƒt at every radius r by pair-wise subtraction of the sedimentation velocity profiles at different time t [20]. r can then be transformed to s*, Equation 6: s* = (1/ω2t) ln(r/rm) The ƒc/ƒt are averaged at each s* to give g(s*) which represents the mass-weighted distribution of sedimentation coefficients. s* is corrected to its standard value at 20 °C in water using the following formula Equation 7: s20,w = s* (ηb /ηw) [(1-ρν)w /(1- ρν)b] where, ν and ρ correspond to the partial specific volume and the density of water at 20 °C, respectively. The value of s0 can be determined by plotting s20,w vs concentration and extrapolating to zero concentration. By placing the calculated values of d0, from DLS, and s0 into the Svedberg equation, the peak molecular weight, Mp, can be calculated as Equation 8: Mp = RT s0 / d0, (1-ρν) BioE 494 – Atomic and Molecular Nanotechnology Fall 2007 Madhu Smitha Harihara Iyer 10 R is the gas constant, T is the absolute temperature, ν is the partial specific volume of the SCK, d0 is the standardized diffusion coefficient, d20,w, extrapolated to zero concentration, and r is the density of the buffer. The anhydrous sphere diameter, D0, of the SCK was then estimated using Equation 9:D0 = (6 Mp ν /πN0)1/3 3.4. Zeta Potential (ζ ) Zeta Potential is the electrokinetic potential difference between the dispersion medium and the stationary layer of fluid attached to the dispersed particle. The value of zeta potential denotes the stability of the colloidal dispersion. Colloids with higher zeta potential are electrically stable as compared to colloids with lower zeta potential which tend to coagulate or flocculate. Calculation of ζ from the measured nanoparticle electrophoretic mobility (µ) employed the Smoluchowski equation Equation 10: µ = ε ζ /η where: ε and η are the dielectric constant and the absolute viscosity of the medium [20]. Figure 5. Zeta Potential Value at different pH Nanoparticle Zeta Potential(mV) PHDCA −48.06 ± 0.20 MePEG2000CA-co- −41.13 ± 0.50 HDCA H2NPEG3400CA- −32.40 ± 2.10 co-HDCA Table 1. Zeta Potential of Nanoparticles Measured BioE 494 – Atomic and Molecular Nanotechnology Fall 2007 Madhu Smitha Harihara Iyer 11 In Table 1, the increase in zeta potential for Poly(H2NPEGCAco-HDCA) at acidic pH, as compared to Poly(hexadecyl cyanoacrylate) and Poly(MePEGCA-co-HDCA) nanoparticles, signifies the protonation of the amino group and their ready availability for folic acid conjugation [14]. Data were acquired in the phase analysis light scattering (PALS) mode following solution equilibration at 25 °C. 3.5. Transmission Electron Microscopy (TEM) Transmission electron microscopy (TEM) is an imaging technique whereby a beam of electrons is transmitted through a specimen, then an image is formed, magnified and directed to appear either on a fluorescent screen or layer of photographic film or to be detected by a sensor. The number-average particle diameter and standard deviations values of the nanoparticles are generated from the TEM analysis of the nanostructures. 3.6. Atomic Force Microscopy (AFM) The atomic force microscope (AFM) is a very high-resolution type of scanning probe microscope, with demonstrated resolution of fractions of a nanometer. The AFM is one of the foremost tools for imaging, measuring and manipulating matter at the nanoscale. The samples are prepared for AFM analysis by depositing a 2-µL drop of the colloidal nanoparticle solution onto freshly cleaved mica and allowing it to dry freely in air. The number-average particle height values and standard deviations are generated from the section analysis. Figure 6. Tapping-mode AFM images 4. Cell internalization of Nano-carriers The effective uptake of folate conjugated nanoparticles by folate receptor mediated endocytosis of the cell can be studied in-vitro. Surface Plasmon Resonance Technology analysis the real time molecular association. Folate binding protein (FBR) are immobilized on the sensor surface of a carboxylated, activated, dextran-coated gold film. Folate conjuagted nanoparticles are introduced to these sensorchips. Free folic acid molecules are used as control. The percenatge of free FBR site to number of folate conjugated nanoparticles estimates the effective internalization of the folate conjugated nanoparticles. The folate-conjugated nanoparticles are known to have 10-fold higher apparent affinity for the FBR than free folate [14]. BioE 494 – Atomic and Molecular Nanotechnology Fall 2007 Madhu Smitha Harihara Iyer 12 Figure 7. Surface Plasmon Resonance Technology In vitro studies of internalization of folate conjugated nanoparticles can be studied by conducting experiments with cancer cell lines namely KB cells, HeLa cells, A549 cells and 4T1cells, which are known to overexpress folate receptors. These cells are cultured in a folate depleted medium to enhance expression of folate binding proteins on their surface and incubated with measured quantity of folate conjuagted nanoparticles loaded with fluorescent dyes like calcein for 4 hrs at 37°C. After incubation, the cells are washed with Lead Sulfide (PbS) solution to remove any unbound and partially bound nanoparticles and analysed using Confocal Laser Scanning Microscopy (CLSM) technique to investigate cell internalizion of the nanoaparticles [4][15]. Figure 8. Confocal microscopic images (light and fluroscent images) of KB cells demonstrating binding and uptake of folate conjuagetd nanoparticles after incubation for (A) 30 min (B) 6 hrs (C) 24 hrs and (D) corresponds to folic acid non conjugated nanoparticles. BioE 494 – Atomic and Molecular Nanotechnology Fall 2007 Madhu Smitha Harihara Iyer 13 Many human tumor cells, including ovarian, lung, breast, endometrial, renal, and colon cancers. are known to express 100 times more folate receptors on their surface as compared to normal cells [7]. This can be experimetnally verified by incubating normal cells and cancer cells with folate receptor conjugated nanoparticles and analysiing their confocal images shown in Figure 9. Figure 9. Confocal images of Normal and HeLa cells incubated in a solution of folate conjugated nanoparticles. Normal cells without abundant folate receprots, show little green fluorescence inside the cells, confirming little uptake of folate conjugated nanoaprticles. HeLa Cells show strong green fluorescence inside cells confirming complete internalization of FA conjugated nanoaprticles 5. MRI Compatible Nanoparticles Super-paramagnetic iron oxide nanoparticles compatible with Magentic Resonance Imaging (MRI) can be synthesized by nanoprecipitation from a solution of ferric and ferrous chloride tetrahydrates dissolved in HCl by addition of NaOH followed by mechanical stirring and ultrasonification [21]. The targetability of these super-paramagnetic iron oxide nanoparticles to cancer cells can be achieved by coating with covalently bound bi-functional polyethylene glycol and folic acid conjugates. Figure 10. Chemical reaction scheme for synthesis of FA-PEG conjugated magnetite nanoparticles BioE 494 – Atomic and Molecular Nanotechnology Fall 2007 Madhu Smitha Harihara Iyer 14 Human cervical carcinoma (HeLa) cell lines which are known to overexpress folate receptors can be used to study the in vitro cellular uptake of the FA conjugated nanoparticles. Dextran coated iron oxide nanoparticles are used as control. Folate receptor positive HeLa cells are incubatd with NP–PEG–FA conjugates, NP–PEG and NP–dextran.The use of NP-dextran as a control is nonspecific contrast enhancement agent for MRI.The uptake of conjugated nanoparticles is evaluated as a function of iron concentration at periodic time intervals in the HeLa cells. HeLa cells cultured with NP-PEG-FA has significantly higher nanoparticle uptake than that of the controls. Figure 11. Cellular uptake of nanoparticles coated with dextran, PEG, or PEG–FA by HeLa cells over time Specific targeting of the NP-PEG-FA to cancerous cells can be evaluated using human osteosarcoma MG-63 cells which have a negative folate receptor cell line and hence reduced probability of uptake of conjugated magnetite nanoparticles. Figure 12. Cellular uptake of NP–PEG–FA conjugates of various concentrations by MG-63 and HeLa cells BioE 494 – Atomic and Molecular Nanotechnology Fall 2007 Madhu Smitha Harihara Iyer 15 Size (Diameter) nm Nano-Sized Drug Carriers 90 80 70 60 50 40 30 20 10 0 Magnetite SWNT G5PAMAM GdNP AuNP Liposome Protein cage SCK Nanoparticles Figure 13. Comparison of sizes of folate conjugated nanoparticles synthesized for drug delivery Relative measure of Nano-Carrier uptake by KB Cells Nanoparticles (10^-9 mg)/ 10^5 cells 2.5 2 1.5 1 0.5 0 Magnetite G5-PAMAM GdNP Liposomes Nanoparticles Figure 14. Comparison of uptake of folate conjugated nanoparticles by KB Cells after 4 hrs of incubation BioE 494 – Atomic and Molecular Nanotechnology Fall 2007 Madhu Smitha Harihara Iyer 16 References [1] Lisa Brannon-Peppas and James O. Blanchette, “Nanoparticle and targeted systems for cancer therapy”, Advanced Drug Delivery Reviews Volume.56, pp.1649– 1659 (2004) [2] CP Leamon, PS Low, “Delivery of macromolecules into living cells: a method that exploits folate receptor endocytosis”, Proceedings of the National Academy of Sciences, Vol.88, Issue.13, pp.5572–5576 (1991) [3] Younsoo Bae, Woo-Dong Jang, Nobuhiro Nishiyama, Shigeto Fukushima and Kazunori Kataoka, “Multifunctional polymeric micelles with folate-mediated cancer cell targeting and pHtriggered drug releasing properties for active intracellular drug delivery”, Molecular Biosystems, Vol.1, pp.242-250 (2005) [4] Antonio Quintana, Ewa Raczka, Lars Piehler, Inhan Lee, Andrzej Myc, Istvan Majoros, Anil K. 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