Abstract:
The present invention relates to a nanoformulated self-assembled pharmaceutical composition for photodynamic therapy. More particularly, the present invention is directed to a self-assembled pharmaceutical composition for photodynamic therapy comprising a photosensitizer, a ligand A which is separated at a specific pH range, and a ligand B of which surface charge changes at a specific pH range and a method for manufacturing the same.

Description:
CLAIM OF PRIORITY 
     This application claims the benefit of Korean Patent Application No. 10-2014-0129040, filed Sep. 26, 2014, the entire contents of which are incorporated herein by reference. 
     FIELD OF THE INVENTION 
     The present invention relates to a nanoformulated self-assembled pharmaceutical composition for photodynamic therapy. More particularly, the present invention is directed to a self-assembled pharmaceutical composition for photodynamic therapy comprising a photosensitizer, a ligand A which is separated at a specific pH range, and a ligand B of which surface charge changes at a specific pH range and a process for preparing the same. 
     BACKGROUND OF THE INVENTION 
     A cancer is caused by uncontrolled growth of cells (neoplasia) and there are over 100 cancer types. Abnormal growth of cells creates a mass of cells (tumor), and this mass of cells penetrates surrounding tissues and spreads to other parts of the body (metastasis). 
     An important matter to consider when administering an effective drug for the treatment of a disease or the diagnosis of the disease region to a human body is how much of the administered effective ingredient reaches the disease region, that is, the problem of drug delivery efficiency. The effective ingredient may reach an unwanted normal tissue and act to cause side effects, and the amount of drug that reaches the disease region may decrease, resulting in the failure of treatment. 
     The drugs that have treatment effects for some diseases show strong toxicity to normal tissues or cells and, therefore, they pose problems of which their side effects or cell toxicity are greater than the treatment effect for the disease. Anti-cancer drugs act on normal tissue cells and have adverse effect on normal tissue cells, rather than removing cancer tissues or having treatment effect. 
     Over 1,300 anti-cancer drugs are known to the public. However, such anti-cancer drugs are often toxic to normal tissue and, thus, have side effects. In addition, since the delivery of anti-cancer drugs to cancer tissues are low due to the characteristics of cancer tissues, the treatment effects are low compared to the side effects. 
     Particularly, due to heterogeneity, that traces and administers drugs by using the affinity the phenomenon that cancer tissue cells produce various type of replicated tumor cells, it is difficult to utilize a method that traces cancer cells and administers drugs by using the affinity between the receptor of cancer cells and the nanoformulated ligands, which is frequently used for nanoformulated drugs. Thus, it is difficult to treat cancers by using the nanoformulated drugs. 
     Furthermore, such heterogeneous extracellular matrix of cancer tissues impedes drug penetration which reduces drug exposure and gradually induces drug-resistance. The cancer tissues having these characteristics are difficult to be treated by targeted therapy using antibody markers due to the heterogeneity of cancer cells. 
     However, due to abnormally rapid division rate of cancer cells and fast growth rate of cancer tissues, cancer tissues are loose and have intercellular cavities therein, compared to normal tissues. In addition, the surface of a cancer tissue includes a number of holes of approximately 100 nm size, has the acidity of less than pH 6.0, and is negatively charged. 
     Tumors in patients generally contain heterogeneous cell populations, and tumor heterogeneity greatly influences the effectiveness of the receptor-ligand targeting strategies that are most popularly used in cancer nanotechnology. In addition, the heterogeneous tumoral extracellular matrix impedes drug penetration which reduces drug exposure and gradually induces drug-resistance. The tumor microenvironment exhibits an increased interstitial fluid pressure caused by leaky vasculature, poor lymphatic drainage, and a high density of cells and their related matrices. 
     Therefore, the penetration of nanoparticle-based drugs is limited to the tumor peripheral region with little diffusion of therapeutic nanoparticles into the tumor interstitial space. The physiological and physical mechanism of drug resistance as a whole is a major cause of the failure of most cancer treatments. Although nanoparticles of small size and multi-functionality are emerging as the next-generation anti-cancer agents, there remain great challenges to the development of ‘smart’ nanoparticles that can specifically respond to tumor-related stimuli in order to overcome the aforementioned tumoral barriers. 
     Self-assembly provides a simple, reproducible and inexpensive way of producing ensembles of nanoparticles with unique plasmonic, photoluminescent, and magnetic properties in a controllable manner. Several stimulus-responsive assembled nanostructures have been thoroughly examined as bio- or chemo-sensors in vitro (Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105, 1547; Cao, Y C.; Jin, R.; Mirkin, C. A. Science 2002, 297, 1536; Zagorovsky, K.; Chan, W. C. W. Angew. Chem., Int. Ed. 2013, 52, 3168; Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757; Pan, Y; Du, X.; Zhao, F.; Xu, B. Chem. Soc. Rev. 2012, 41, 2912). However, thus far, these types of “smart” ensembles have rarely been investigated in vivo owing primarily to these inherent physiological obstacles. One commonality among tumors is acidity; the microenvironment usually has a pH of ˜6.8 and endo/lysosomes experience even lower pH values of 5.0-5.5. 
     Magnetic resonance imaging (MRI) is the most effective imaging diagnosis equipment up to date that can form images of the organs of living humans or animals in a non-invasive way in real time. MRI contrasting agent helps each tissue and blood vessel to be shown more clearly, so that more precise diagnosis is possible. 
     There are two types of MRI contrasting agent: T 1 , which makes the target site brighter and T 2 , which makes it darker. Paramagnetic gadolinium complex is widely used as T 1  contrasting agent, but low molecular weight of gadolinium complex makes the residence time within the blood vessel and body short, making exact diagnosis of vessel disease more difficult, and there has been a report that it may cause systemic fibrosis in a person with degraded kidney function. 
     Photodynamic therapy is a treatment that uses a photosensitizer which causes chemical reactions along with light and oxygen to selectively kill cells of a tissue in need of treatment. Unlike general anti-cancer drugs or radiation therapies which have an effect on normal tissue cells, photodynamic therapy has the advantage of greatly reducing the side effects of the anti-cancer drugs or radiation therapies, due to targeting and killing the cells of diseased tissues and selectively killing them. 
     The object of the present invention is to provide a pharmaceutical composition for photodynamic therapy, of which particle size is smaller than below 100 nm-sized holes on cancer tissue surface, wherein the surface charge of the composition changes from negative to positive when the composition reaches cancer tissues, thereby allowing for approach and penetration of the composition to the cancer tissues, and wherein the photosensitizer can selectively and effectively penetrate to the cancer tissues due to a linker which separates at an acidic condition of the cancer tissues, and thereby effectively killing cancer tissue cells by forming a large amount of reactive oxygen species activated by light and the photosensitizer. 
     SUMMARY OF THE INVENTION 
     Technical Problem 
     The primary object of the present invention is to provide a self-assembled pharmaceutical composition for photodynamic therapy comprising a photosensitizer, a ligand A which is separated at a specific pH range, and a ligand B of which surface charge changes at a specific pH range. 
     Another object of the present invention is to provide a process for preparing self-assembled pharmaceutical composition for photodynamic therapy, comprising: (i) preparing the ligand composition by adding solution comprising a ligand A which is separated at a specific pH range, and a ligand B of which surface charge changes at a specific pH range to chloroform; and (ii) separating the nanoformulated pharmaceutical composition for photodynamic therapy from the ligand composition. 
     Technical Solution 
     The primary object of the present invention can be accomplished by providing a self-assembled pharmaceutical composition for photodynamic therapy comprising a photosensitizer, a ligand A which is separated at a specific pH range, and a ligand B of which surface charge changes at a specific pH range. 
     The photosensitizer may be chlorin E6. Also, the photosensitizer may be bonded to the ligand A or ligand B. 
     The ligand A may contain PEG, imidazole group, catechol and chlorin E6 (Ce6). The hydrophilicity of the PEG helps the ligand composition retain its form; the imidazole group makes the ligand composition change its form in response to pH change; the catechol bonds to extremely small iron oxide nanoparticles (ESIONs); and the Ce6 emits reactive oxygen species (ROS) that destroy cancer cells when exposed to a proper light. 
     Furthermore, the ligand B may contain PEG, imidazole group, phenyl group and Ce6. The hydrophilicity of the PEG helps the ligand composition retain its form; the imidazole group makes the ligand composition change its form in response to pH change; the lipophilicity of the phenyl group helps the ligand composition retain its form; and the Ce6 emits reactive oxygen species (ROS) that destroy cancer cells when exposed to a proper light. 
     In an embodiment of the self-assembled pharmaceutical composition of the present invention, the ligand A may be a compound of formula (I), as shown in  FIG. 19 , wherein n and m are independently 5-500. Also, the imidazole group of the formula (I) may be substituted by triazole or piperazine group. 
     Furthermore, in an embodiment of the self-assembled pharmaceutical composition of the present invention, the ligand B may be a compound of formula (II), as shown in  FIG. 20 , wherein n and m are independently 5-500. Also, the imidazole group of the formula (II) may be substituted by triazole or piperazine. In addition, the phenyl group of the formula (II) may be substituted by naphthyl or indole group. 
     In one embodiment of the self-assembled pharmaceutical composition of the present invention, the pH range at which ligand A separates or the surface charge of ligand B changes may be 4-7.2. 
     In one embodiment of the self-assembled pharmaceutical composition of the present invention, the composition may further comprise an MRI contrasting nanoparticle. The MRI contrasting nanoparticle may be an iron oxide nanoparticle. Furthermore, the size of the iron oxide nanoparticle may be 1 nm-100 nm. 
     Another object of the present invention can be achieved by providing a process for preparing self-assembled pharmaceutical composition for photodynamic therapy, comprising: (i) preparing the ligand composition by adding to chloroform a mixture solution which contains a ligand A which is separated at a specific pH range, and a ligand B of which surface charge changes at a specific pH range; and (ii) separating the nanoformulated pharmaceutical composition for photodynamic therapy from the ligand composition. 
     In an embodiment of the process for preparing self-assembled pharmaceutical composition of the present invention, the photosensitizer may be chlorin E6. Also, the photosensitizer may be bonded to the ligand A and ligand B. 
     In one embodiment of the process for preparing self-assembled pharmaceutical composition of the present invention, the ligand A may be a compound of formula (I) and the ligand B may be a compound of formula (II). 
     In one embodiment of the process for preparing self-assembled pharmaceutical composition of the present invention, the pH range at which ligand A separates or the surface charge of ligand B changes may be 4-7.2. 
     In one embodiment of the process for preparing self-assembled pharmaceutical composition of the present invention, the solvent of the mixture solution may be DMSO (dimethyl sulfoxide), DMF (dimethylformamide) or THF (tetrahydrofuran). Also, the organic solvent may be chloroform, dichloromethane or 1,2-dichloroethane. 
     In one embodiment of the process for preparing self-assembled pharmaceutical composition of the present invention, the ligand A may be produced by a method comprising: (i) preparing poly(ethylene glycol)-poly(β-benzyl-L-aspartate) (PEG-PBLA) by polymerizing β-benzyl-L-aspartate N-carboxy anhydride (BLA-NCA) in the mixture of DMF and CH 2 Cl 2 ; (ii) preparing platform ligand by bonding chlorin E6 (Ce6) to the PEG-PBLA; and (iii) preparing ligand A by the aminolysis of the platform ligand using 1-(3-amynopropyle)imidazole and dopamine. 
     Furthermore, in an embodiment of the process for preparing self-assembled pharmaceutical composition of the present invention, the ligand B may be produced by a method comprising: (i) preparing poly(ethylene glycol)-poly(β-benzyl-L-aspartate) (PEG-PBLA) by polymerizing β-benzyl-L-aspartate N-carboxy anhydride (BLA-NCA) in the mixture of DMF and CH 2 Cl 2 ; (ii) preparing platform ligand by bonding chlorin E6 (Ce6) to the PEG-PBLA; and (iii) preparing ligand A by the aminolysis of the platform ligand using 1-(3-amynopropyle)imidazole and 3-phenyl-1-propylamine. 
     In one embodiment of the process for preparing self-assembled pharmaceutical composition of the present invention, the composition may further comprise mixing the ligand composition of the step (i) with an MRI contrasting nanoparticle. 
     In one embodiment of the process for preparing self-assembled pharmaceutical composition of the present invention, MRI contrasting nanoparticle may be iron oxide nanoparticle. Also, the size of the iron oxide nanoparticle may be 1 nm to 100 nm. 
     Advantageous Effects 
     The nanoformulated pharmaceutical composition for photodynamic therapy of the present invention is a self-assembled nanostructure that reacts to the acidic stimulation of tumors and functions to deliver the pharmaceutical composition selectively to cancer cells. Therefore, the nanoformulated pharmaceutical composition for photodynamic therapy of the present invention allows for effective photodynamic therapy of heterogeneous and drug-resistant tumor on early stages. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. 
         FIG. 1  shows H-NMR analysis of the platform ligand (PEG-PBLA-Ce6) synthesized in Example 1 of the present invention. 
         FIG. 2  shows H-NMR analysis of the ligand A (PEG-p(API &amp; DOPA- L -Asp)-Ce6) synthesized in Example 1 of the present invention. 
         FIG. 3  shows H-NMR analysis of the ligand B (PEG-p(API &amp; PPA- L -Asp)-Ce6) synthesized in Example 1 of the present invention. 
         FIG. 4  shows GPC curves of the platform ligand, ligand A and ligand B synthesized in Example 1 of the present invention. 
         FIG. 5 a    shows pH-dependent structural transformation change in pH-sensitive magnetic nanoformulation (PMN): self-assembly of iron oxide nanoparticles and pH-responsive ligands (inset of (a): TEM images of PMNs at pH 7.4, 6.8, and pH 5.5, DLS measurement of PMN colloidal dispersion at pH 7.4 showing an average diameter of ˜70 nm). 
         FIG. 5 b    shows schematic representation of pH-dependent structural transformation and related magnetic/photoactivity change in PMNs. 
         FIG. 5 c    shows zeta-potential and DLS size measurement of PMNs (0.1 mg/mL) as a function of pH following a 1 h incubation (n=3) (DLS measurements were conducted using a material refractive index of 2.42, solvent refractive index of 1.33 (water) and a viscosity of 0.8872). 
         FIG. 6 a    shows size distribution by number. 
         FIG. 6 b    shows size distribution by intensity. 
         FIG. 6 c    shows raw correlation data of InS-NP colloidal dispersion at pH 7.4. 
         FIG. 7 a    shows transmittance of PMN suspension measured at selected pH values (the inset presents data for pH-dependent turbidity of the PMN solution with a transition pH value of about 6.0. The pH was gradually changed from 4.0 to 7.5 (●) and from pH 7.5 to 4.0 (∘)). 
         FIG. 7 b    shows pH-dependent fluorescence intensity and near-infrared fluorescence imaging of (1) self-assembled ligands and (2) PMNs. 
         FIG. 7 c    shows pH-dependent singlet oxygen generation of (1) self-assembled ligands and (2) PMNs. 
         FIG. 7 d    shows optical, fluorescence, and MR phantom images (examined in a clinical 1.5 T MRI scanner) of a concentration gradient of PMNs at three different pH values. 
         FIG. 7 e    shows pH-dependent magnetic relaxation properties of PMNs. 
         FIG. 8  shows the XRD patterns of PMNs and 3 nm-sized ESIONs (extremely small iron oxide nanoparticles). 
         FIG. 9 a    shows the magnetic properties of PMNs through an M-H curve between the magnetic field of 20 kOe and −20 kOe, and 
         FIG. 9 b    shows the magnetic properties of PMNs through an M-T curve for PMNs under 100 Oe with the temperature varying from 300 K to 4 K. 
         FIG. 10  shows MR phantom images of a concentration gradient of InS-NPs at three different pH values. 
         FIG. 11 a    shows cellular uptake of PMNs (equivalent to 1 μg/mL Ce6) for HCT116 cancer cells at pH 7.4 or 6.8 (4 h incubation) analyzed by flow cytometry (the inset shows near-infrared fluorescence imaging of HCT116 cancer cells treated with different concentrations of PMNs at pH 7.4 and 6.8 (4 h incubation)). 
         FIG. 11 b    shows confocal microscopy images of activated PMNs in HCT116 cancer cells (the endosomes/lysosomes were stained with Lysotracker Green which selectively labels lysosomes). 
         FIG. 11 c    shows BioTEM images of PMN uptake by HCT116 cancer cells. 
         FIG. 11 d    shows MTT assays of HCT116 cells exposed to PMNs and free Ce6 without laser irradiation. 
         FIG. 11 e    shows MTT assays of HCT116 cells exposed to PMNs and free Ce6 with laser irradiation. 
         FIG. 11 f    shows fluorescence (upper) and MR (lower) images of HCT116 cancer cell pellets (1×10 6  cells) incubated with various concentrations of PMNs (0, 12.5, 25, or 50 μg Fe/mL) for 4 hours. 
         FIG. 12 a    shows in vivo T 1 -weighted MR images and color-mapped images of tumor sites before and 1 or 2 hours after intravenous injection of InS-NPs into nude mice bearing HCT116 tumors (the dashed regions indicate tumor site) (the dashed regions indicates tumor sites). 
         FIG. 12 b    shows in vivo T 1 -weighted MR images and color-mapped images of tumor sites before and 1 or 2 hours after intravenous injection of PMNs into nude mice bearing HCT116 tumors (the dashed regions indicate tumor site) (the dashed regions indicates tumor sites). 
         FIG. 12 c    shows plot of signal intensity enhancement (ROI i /ROI 0 ) versus time after injection of PMNs and InS-NPs. 
         FIG. 12 d    shows blood circulation data (plasma iron concentration vs time) for PMNs and InS-NPs in nude mice (see inset; n=3 for each group). 
         FIG. 12 e    shows inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis of tumor tissue showing &gt;2-fold increase of PMNs than InS-NPs in HCT116 tumors at 12 h after intravenous injection. 
         FIG. 12 f    shows in vivo NIR imaging of nude mice bearing HCT116 tumors after intravenous injection of PMNs, InS-NPs or free Ce6 (equivalent to 0.2 mg/kg Ce6) (the dashed region indicates tumor site). 
         FIG. 12 g    shows CLSM shows Ce6 uptake by tumor cells in tumor tissue of nude mice after intravenous injection of PMNs. 
         FIG. 13 a    shows fluorescence images of organs extracted 24 hours post-treatment. 
         FIG. 13 b    shows measured signal intensities of organs extracted 24 hours post-treatment. 
         FIG. 14 a    is a photograph showing a PMN-based targeted photodynamic therapy (PDT). 
         FIG. 14 b    is a schematic illustration and comparison of PDT efficacy of PMNs and InS-NPs in homogeneous and heterogeneous tumors. 
         FIG. 14 c    shows immunohistochemical staining of heterogeneous tumors for SDF-1, P-gp and CD31. 
         FIG. 14 d    shows images of mice bearing homogeneous HCT116 tumors before and after PDT activation (upper), and Hematoxylin and eosin (H&amp;E) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining of tumor tissue sections in order to determine treatment effectiveness in terms of tumor cell death by apoptosis (below). 
         FIG. 14 e    shows homogeneous HCT116 tumor volumes in the four treatment groups after treatment. 
         FIG. 14 f    shows images of mice bearing heterogeneous tumors before and after PDT activation (upper), and H&amp;E and TUNEL staining of tumor tissue sections in order to determine treatment effectiveness in terms of tumor cell death by apoptosis (below). 
         FIG. 14 g    shows heterogeneous tumor (solid dots) volumes in the four treatment groups after treatment. 
         FIG. 15  shows body weight measurements throughout the PDT treatments for HCT116 tumor-bearing mice ( FIG. 15 a   ), and heterogenetic CT26 tumor-bearing mice ( FIG. 15 b   ). 
         FIG. 16 a    shows serum biochemistry results for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and D-lactate dehydrogenase (D-LDH) from nude mice (n=4) administered with PMNs or PBS buffer. 
         FIG. 16 b    shows histological examination images of heart, kidney, liver, lung and spleen stained with haematoxylin and eosin (H&amp;E) (100×). 
         FIG. 17  shows the PMNs were fabricated by co-assembly of ESIONs, ligand A and ligand B. 
         FIG. 18  shows self-assembly of ESIONs into colloidal magneto-core shell structures. 
         FIG. 19  shows an embodiment of ligand A. 
         FIG. 20  shows an embodiment of ligand B. 
         FIG. 21  shows the template for synthesis of ligands A and B. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Hereinafter, the present invention will be described in greater detail with reference to the following examples. The examples are given only for illustration of the present invention and not to be limiting the present invention. 
     Example 1. Synthesis of Ligands 
     As shown in  FIG. 21 , ligands A and B were synthesized by using poly(ethylene glycol)-poly(β-benzyl-L-aspartate) (PEG-PBLA) as a template. 
     In order to prepare PEG-PBLA, β-Benzyl-L-aspartate N-carboxy anhydride (BLA-NCA) (3 g, 12 mmol) was polymerized in a mixture of DMF (20 mL) and CH 2 Cl 2  (50 mL) at 40° C. by initiation from the terminal primary amino group of α-methoxy-ω-amino-poly(ethylene glycol) (MW=2,000 Da, 240 mg, 120 μmol). PEG-PBLA was purified by precipitation in ether (3 L) three times. In order to synthesize PEG-PBLA-Ce6 (platform ligand), Ce6 was attached to the amine groups of PEG-PBLA via the conventional carbodiimide reaction. The PEG-PBLA (0.5 g, 32.5 μmol,) and a mixture of Ce6 (23.4 mg, 39.0 μmol), dicyclohexylcarbodiimide (9.6 mg, 46.8 μmol), and N-hydroxysuccinimide (5.4 mg, 46.8 μmol) were dissolved separately in DMSO (5 mL) and the solutions were stirred thoroughly for 3 h prior to the condensation reaction. The two reactant solutions were then mixed and stirred at room temperature. After 24 h, the reaction mixture was filtered to remove the insoluble by-products (e.g., dicyclohexylurea) and dialyzed against deionized water for 2 days (Spectra/Por; molecular weight cutoff size, MWCO: 1,000 Da). The final solution was lyophilized to obtain the platform ligand. The hydrogen nuclear resonance spectroscopy (“H-NMR”) analysis of the platform ligand (PEG-PBLA-Ce6) at 300 MHz with DMSO-d 6  is illustrated in  FIG. 1 , where δ=9.7 ppm (1H, s, —CH═ of Ce6), 8.1 ppm (1H, s, —N H —), 7.3 ppm (5H, s, —CH 2 C 6   H   5 —), 5.0 ppm (2H, —C H   2 C 6 H 5 —), 4.6 ppm (1H, m, —NHC H C═O—), 3.5 ppm (182H, s, —C H   2 C H   2 O— of mPEG), 3.2 ppm (3H, s, C H   3 — of mPEG) and 2.9-2.5 ppm (2H, m, CHC H   2 C═O). 
     Ligand A was synthesized via aminolysis of the platform ligand with 1-(3-aminopropyl)imidazole (API) and dopamine. PEG-PBLA-Ce6 (0.5 g, 18.5 μmol) was dissolved in DMSO (5 mL), followed by the reaction with dopamine (0.1 g, 0.7 mmol) under nitrogen atmosphere at 25° C. for 1 h. Then, API (0.5 g, 3.9 mmol) was added under nitrogen at 25° C. and stirred for 4 h. The reaction mixture was added dropwise into a cooled aqueous solution of 0.1 N HCl (20 mL) and dialyzed against a 0.01 N HCl solution three times (Spectra/Por; MWCO: 1,000 Da). The final solution was lyophilized to obtain ligand A. The H-NMR analysis of ligand A (PEG-p(API &amp; DOPA- L -Asp)-Ce6) at 300 MHz with DMSO-d 6  is illustrated in  FIG. 2 , where δ=7.8 ppm (1H, s, —NC H ═N— of imidazole ring), 7.7 ppm (1H, s, —NC H ═CH— of imidazole ring), 7.3 ppm (1H, s, —CHC H ═N— of imidazole ring), 6.6-6.5 ppm (1H, m, —CH 2 ═C H CH— and —CH 2 C H ═CH— of dopa), 6.4 ppm (1H, m, ═C H CO— of dopa), 4.5 ppm (1H, m, —NHC H C═O—), 4.2 ppm (2H, m, ═NC H   2 CH 2 ), 3.5 ppm (182H, s, —C H   2 C H   2 O— of mPEG chain), 3.0 ppm (2H, m, —NHC H   2 CH 2 —), and 2.8-2.5 ppm (2H, m, CHC H   2 C═O). 
     Ligand B was synthesized via aminolysis of the platform ligand with API and 3-phenyl-1-propylamine (PPA). PEG-PBLA-Ce6 (0.5 g, 18.5 μmol) was dissolved in DMSO (5 mL), followed by reacting with PPA (0.1 g, 0.9 mmol) under nitrogen at 25° C. for 1 h. Then, API (0.5 g, 3.9 mmol) was added under nitrogen at 25° C. and stirred for 4 h. After the reaction, the mixture was added dropwise into a cooled 0.1 N HCl solution (20 mL) and dialyzed against an aqueous solution of 0.01 N HCl three times (MWCO: 1,000 Da). The final solution was lyophilized to obtain ligand B. The H-NMR analysis of ligand B (PEG-p(API &amp; PPA- L -Asp)-Ce6) 300 MHz with DMSO-d 6  is illustrated in  FIG. 3 , where δ=7.8 ppm (1H, s, —NC H ═N— of imidazole ring), 7.7 ppm (1H, s, —NC H ═CH— of imidazole ring), 7.3 ppm (1H, s, —CHC H ═N— of imidazole ring), 7.3-7.2 (5H, m, —CH 2 C 6   H   5 — of PPA), 4.5 ppm (1H, m, —NHC H C═O—), 4.2 ppm (2H, m, ═NC H   2 CH2), 3.5 ppm (182H, s, —C H   2 C H   2 O— of mPEG chain), 3.0 ppm (2H, m, —NHC H   2 CH 2 —), and δ2.8-2.5 ppm (2H, m, CHC H   2 C═O. 
     In the GPC measurements ( FIG. 4 ), all polymeric ligands showed unimodal molecular weight distribution indicating no homopolymer residues in the copolymer products. The Ce6 content of the ligand was examined by the fluorescence spectroscopy (λ ex =650 nm and λ em =675 nm, Table 1). 
     
       
         
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Structural characteristics of platform ligand, ligand A and ligand B. 
               
             
          
           
               
                   
                   
                 DP 
                 DS 
                 DS 
                 DS 
                 DS 
                   
                   
               
               
                 Code 
                 Copolymer 
                 (BLA) a   
                 (API) b   
                 (PPA) c   
                 (Dopa) d   
                 (Ce6) e   
                 PDI f   
                 M n   g   
               
               
                   
               
               
                 Platform 
                 PEG 1 -PBLA 60 -Ce6 1   
                 60 
                 — 
                 — 
                 — 
                 1 
                 1.11 
                 16,000 
               
               
                 Ligand 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Ligand A 
                 PEG 1 -p(API 55  &amp; 
                 60 
                 55 
                 — 
                 5 
                 1 
                 1.14 
                 17,200 
               
               
                   
                 Dopa 5 -L-Asp) 60 -Ce6 1   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Ligand B 
                 PEG 1 -p(API 50  &amp; 
                 60 
                 50 
                 10 
                 — 
                 1 
                 1.09 
                 16,100 
               
               
                   
                 PPA 10 -L-Asp) 60 -Ce6 1   
               
               
                   
               
               
                   a Degree of polymerization of BLA on the basis of the  1 H-NMR results. 
               
               
                   b Degree of substitution of API on the basis of the  1 H-NMR results. 
               
               
                   c Degree of substitution of PPA on the basis of the  1 H-NMR results. 
               
               
                   d Degree of substitution of Dopa on the basis of the  1 H-NMR results. 
               
               
                   e Degree of substitution of Ce6 on the basis of the fluorescence spectroscopy. 
               
               
                   f Number-averaged (M n ), weight-averaged molecular weight (M w ) and polydispersity index (PDI = M w /M n ) were determined by GPC 
               
               
                   g As determined by  1 H-NMR. 
               
             
          
         
       
     
     Example 2. Synthesis of ESIONs 
     ESIONs were synthesized via thermal decomposition of iron-oleate complex in the presence of oleyl alcohol using the previously reported method (Kim, B. H.; Lee, N.; Kim, H.; An, K.; Park, Y. I.; Choi, Y.; Shin, K.; Lee, Y.; Kwon, S. G.; Na, H. B.; Park, J. G.; Ahn, T. Y.; Kim, Y. W.; Moon, W. K.; Choi, S. H.; Hyeon, T.  J. Am. Chem. Soc.  2011, 133, 12624). Briefly, 1.8 g of iron-oleate complex (2 mmol), 0.57 g of oleic acid (2 mmol), and 1.61 g of oleyl alcohol (6 mmol) were dissolved in 10 g of diphenyl ether at room temperature. The mixture was heated to 250° C. at a constant heating rate of 10° C./min and then kept at this temperature for 30 min under inert atmosphere. As the reaction proceeded, the initial brown transparent solution turned black. After the reaction, the mixture containing nanoparticles was removed from the heater and allowed to cool to room temperature and, then, 50 mL of acetone was added to precipitate the nanoparticles. The nanoparticles were pelleted by centrifuging at 40,000 rpm for 4 hours, the supernatant decanted and the nanoparticles redispersed in n-hexane or chloroform. 
     Example 3. Fabrication of PMNs 
     For self-assembly, a solution prepared by mixing 15 mg of Ligand A and 15 mg of Ligand B in 3 mL of DMSO was added slowly to 5 mL of colloidal ESIONs (0.4 mg Fe/mL) in chloroform. The mixture was incubated on a shaker at room temperature for 30 min. Chloroform was then completely removed by evaporation under vacuum and deionized water was added to the colloidal suspension in DMSO to reach a total volume of 5 mL. The DMSO was completely substituted with deionized water using a dialysis membrane (Spectra/Por; MWCO: 12,000 Da). Excess ligands were removed by centrifugation and washed 3-5 times with spin filter (Millipore, MWCO: 100,000 Da, 10,000×g, for 10 min) The resulting nanoparticles were re-dispersed in water. 
     Example 4. Fabrication of Self-Assembled Ligands 
     A mixed solution of 15 mg of Ligand A and 15 mg of Ligand B in 3 mL of DMSO was added slowly to 5 mL of chloroform. The mixture was incubated on a shaker at room temperature for 30 min. Then, chloroform was removed completely by evaporation under vacuum. Thereafter, deionized water was added to the colloidal solution in DMSO to reach a total volume of 5 mL. DMSO was completely substituted with deionized water using a dialysis membrane (Spectra/Por; MWCO, 12,000 Da). Excess ligands were removed by centrifugation or washed with spin filter (Millipore, MWCO: 100,000 Da, 10,000×g, for 10 min) 3-5 times. The resulting nanoparticles were re-dispersed in water. 
     Example 5. Fabrication of pH-Insensitive Nanoparticle Assembles (InS-NPs) 
     A solution prepared by mixing 30 mg of platform ligand in 3 mL DMSO was added slowly to 5 mL of a colloidal ESIONs (0.4 mg Fe/mL) suspension in chloroform. The mixture was incubated on a shaker at room temperature for 30 min. Then, chloroform was removed completely by evaporation under vacuum. Thereafter, deionized water was added to the colloidal solution in DMSO to reach a total volume of 5 mL. DMSO was completely substituted with deionized water using a dialysis membrane (Spectra/Por; MWCO, 12,000 Da). Excess ligands were removed by centrifugation or washed with spin filter (Millipore, MW, 100,000 Da, 10000×g, for 10 min) 3-5 times. The resulting nanoparticles were re-dispersed in water. 
     Example 6. Characterizations of Materials 
     TEM measurements were performed on a JEOL EM-2010 microscope operated at 200 kV. The powder X-ray diffraction patterns were obtained with a Rigaku D/Max-3C diffractometer equipped with a rotating anode and a Cu Kα radiation source (λ=0.15418 nm). The present inventors performed elemental analysis by inductively coupled plasma atomic emission spectroscopy (ICP-AES) using an ICPS-7500 spectrometer (Shimadzu) and inductively coupled plasma-optical emission spectrometer (ICP-OES) (Perkin-Elmer Optima 4300 DV). UV/visible absorption spectra were collected on UV-2450 spectrophotometer (Shimadzu, Japan). Particle size and zeta potential were measured with Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). For the analysis of size, the following parameters, material refractive index (2.42), material absorbance (0.2), dispersant refractive index (1.33), and dispersant viscosity (0.8872) were used. The magnetic properties were investigated using a superconducting quantum interface device (SQUID) magnetometer (Quantum Design MPMS XL). Hydrodynamic sizes were obtained using dynamic light scattering (DLS) (Zetasizer Nano ZS (Malvern Instruments, Malvern, UK)) instrument at 25° C. 
     Example 7. Measurements of Critical Aggregation Concentration (CAC) 
     A stock solution of Hoechst 33342 (1.4×10 −3  M) in double-distilled water was prepared and stored at 4° C. The Hoechst 33342 solution was mixed with solutions containing the samples at the concentrations of 1.0×10 −3  mg/mL to 1.0 mg/mL. The final concentration of Hoechst 33342 in each sample solution was 7.0×10 −4  M. The resultant fluorescence was measured on a RF-5301PC (Shimadzu, Japan) with λ ex =355 nm and λ em =457 nm and the slit widths were ex=3 nm and em=3 nm, respectively. 
     Example 8. Transmittance Measurement of PMNs at Different pH 
     The light transmittance measurements of PMN solutions (2 mg/mL, without Ce6 attachment) were obtained using a UV-Visible spectrophotometer at 500 nm while the pH value of solution was gradually decreased from 7.5 to 4.0 by adding 0.1 N HCl, and increased from 4.0 to 7.5 by adding 0.1 N NaOH solution. 
     Example 9. pH-Dependent Fluorescence Intensity Measurement 
     The fluorescence intensity of PMNs or self-assembled ligands (2 mg/mL, 650 nm excitation and 675 nm emission) was measured using a fluorescence plate reader (Tecan Genios, Durham, N.C.), while the pH of the solution was gradually decreased from 7.5 to 4.0 by adding 0.1 N HCl solution. 
     Example 10. pH-Dependent Singlet Oxygen Generation (SOG) Measurement 
     In order to evaluate the SOG, samples (2 mg/mL, 100 μL) at different pH were mixed with singlet oxygen sensor green (SOSG, 2.0 μM, 100 μL). SOG was induced by irradiation with a 670 nm laser source (Institute of Electronics) at 5 mW/cm 2  intensity for 4 min. SOSG fluorescence was detected (λ ex =494 nm and λ em =534 nm) after irradiation to determine the SOG of the samples. SOG was evaluated by SOSG fluorescence enhancement compared to the background. 
     Example 11. Cell Culture 
     HCT116 (human colon cancer, KCLB No. 10247) cells, CT26 (murine colorectal carcinoma cell, KCLB No. 80009) and M2-10B4 (murine bone marrow stromal cell, KCLB No. 21972) were obtained from the Korean Cell Line Bank. HCT116 cells were cultured in 10 mL of Roswell Park Memorial Institute (RPMI-1640) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. CT26 cells were cultured in Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin. M2-10B4 was cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. Culturing conditions for all cells were 37° C. in 100% humidity and 5% CO 2 . Drug resistant CT26 cells (CT26/MDR) were developed by exposing CT26 cells to increasing doses (1, 5, 10, 25, 50, 100, 500, 1,000, and 5,000 ng/mL) of DOX for 24 h followed by 3-4 days of recovery before exposing to the next dose. CT26/MDR was then frozen and stored in liquid nitrogen. Fresh aliquots of CT26/MDR were used in all experiments to ensure that they did not revert to a drug sensitive phenotype. 
     Example 12. pH-Dependent Cell Uptake 
     HCT116 cells were exposed to PMNs, InS-NPs and Ce6 at different pHs to verify their cellular uptake respectively. The cells were incubated for 2 h, washed, harvested, and re-suspended with DPBS. In vitro cellular uptake was quantified using a flow cytometer (Beckman, San Jose, Calif., USA). For each sample 10,000 cells (gated events) were counted, and free Ce6 fluorescence was detected with logarithmic settings (FL4; λ em =670 nm). Cells were counted as positive if their fluorescence (FL4) was higher than that of cells from an untreated cell suspension. Each experiment was analyzed statistically using the CXP Analysis Program. 
     Example 13. Confocal Laser Scanning Microscope 
     Confocal laser scanning microscope (LSM 510 Meta; Zeiss, Germany) was used to carry out fluorescence imaging. An optimal pinhole size of 120 μm was selected to exclude fluorescent light emitted from out-of-focus planes above and below the focusing plane. The excitation/emission wavelengths were 340/488 nm for DAPI, 490/520 nm for FITC, 555/578 nm for RITC, and 650/670 nm for Ce6. Fluorescence images were analyzed using LSM Image Browser software (Zeiss). 
     Example 14. Cell MR Imaging 
     In order to label the cells with PMNs, HCT116 cells were seeded onto culture dishes in 10 mL of media and grown overnight. Subsequently, PMNs of 0, 12.5, 25, and 50 μg/mL were added. After 4 h, the cells were washed twice with PBS and detached by adding 1 mL of trypsin/EDTA. After centrifugation, cells were dispersed in culture media and transferred to a 1.5 mL test tube. Cell pellets were prepared by centrifugation at 1,000 rpm for 5 min. T 1 -weighted MR images were acquired with a head coil on a 1.5 T MR scanner (Signa Excite, GE Healthcare). 
     Example 15. Pharmacokinetic Analysis 
     For the plasma concentration-time experiment, the mice were injected with PMNs and InS-NPs respectively (2 mg Fe/kg) via the tail vein. Blood was collected at 1 min, 30 min, 1 h, 3 h, 8 h and 20 h after the injection. Plasma was isolated from red blood cells by centrifugation at 1,000 rpm for 10 min. The plasma (100 μL) for each blood sample was subsequently mixed with 70% nitric acid (1 mL) at room temperature for 12 h followed by centrifugation (10,000 rpm for 5 min), and the supernatant was used for inductively coupled plasma optical emission spectrometer analysis (ICP-OES, Optima 4300 DV, Perkin-Elmer) after 5-fold dilution with 2% nitric acid. Fe uptake in the tumor was measured 12 h after PMNs or InS-NPs injection (2 mg Fe/kg). Dissected tumor tissues were weighed, homogenized, and treated with scintillation mixtures. A volume of 60% nitric acid was added to each sample and tissue was incubated for 24 h at 60° C. after which the solutions were centrifuged at 13,000 rpm for 30 min and the supernatant was diluted 10-fold with 2% nitric acid. Determination of Fe uptake content in tumor was performed by ICP-OES, and the nanoparticle uptake in tumor was calculated as gram of Fe content per gram of tissue (g Fe/g Tissue). 
     Example 16. Immunohistochemistry 
     Tumor tissues were fixed in 10% neutral buffered formalin and frozen sectioned into 5 micron thick slices. SDF-1 and P-gp were immunolabeled with anti-SDF-1 antibody (abcam) and anti-P-glycoprotein antibody (abeam), then the samples were incubated for 60 min at ambient temperature with RITC (λ ex /λ em , 555/578 nm) or FITC (λ ex /λ em , 490/520 nm) conjugated secondary antibody for further confocal laser scanning microscope analysis. 
     Example 17. Tumor Histology 
     For histology analysis, tumor tissues from control and treated mice were fixed in 10% neutral buffered formalin and frozen sectioned into 5 micron thick slices, stained with hematoxylin &amp; eosin (H&amp;E) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), and were examined by a digital microscope (Leica QWin) and a confocal laser scanning microscope (LSM 510 Meta; Zeiss, Germany). 
     Example 18. In Vivo Toxicity Evaluation of PMNs 
     Healthy nude mice were intravenously injected with a suspension of PMNs or normal saline (control group). After 2 weeks, the mice were sacrificed and major viscera collected. The heart, liver, spleen, kidney, and lung were stained with H&amp;E. Reagent kits of ALT, AST, ALP and D-LDH were employed to analyze the serum which was isolated from blood sampled by eyeball extirpation. 
     Example 19. In Vitro Cellular Uptake 
     The tumor cellular uptake of PMNs was monitored at different pH conditions (pH 7.4 and 6.8) using a KODAK In vivo Image Station (Image Station 4000 MM; Kodak, New Haven, Conn., USA) and a flow cytometer (Beckman, San Jose, Calif., USA). HCT116 cells (1×10 5  cells/mL) were incubated with each sample for 2 h in RPMI-1640 medium (pH 7.4 or 6.8, with 1% penicillin-streptomycin) at 37° C. and then analyzed. To ameliorate photobleaching during microscope observation, a drop of anti-fade mounting solution (5% N-propyl gallate, 47.5% glycerol, and 47.5% Tris-HCl, pH 8.4) was added to the cells. 
     Example 20. In Vivo Studies 
     All in vivo studies conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, USA (NIH publication no. 85-23, 1985, revised 1996), and mice were maintained under the guidelines of an approved protocol from the Institutional Animal Care and Use Committee (IACUC) of the Catholic University of Korea (Republic of Korea). 
     Example 21. Characterization of pH-Sensitive Magnetic Nanogrenades (PMNs) 
     Ce6 grafted poly(ethylene glycol)-poly(β-benzyl-L-aspartate) (PEG-PBLA-Ce6) was synthesized to provide a platform ligand in which the flanking benzyl ester groups readily react with primary amines via nucleophilic attack. Imidazole (pKa, ˜6.8) was then easily incorporated as an ionizable group to impart pH sensitivity to the tumor microenvironment. On the basis of this platform, the present inventors further engineered two ligand derivatives: ligands A and B. For ligand A, catechol groups were added to facilitate self-assembly as they can act as high-affinity anchors for iron oxide nanoparticles. In contrast, the hydrophobicity of ligand B was tuned using 3-phenyl-1-propylamine to produce a critical phase transition of PMNs that is activatable by tumor endo/lysosomal pH of ˜5.5. The PMNs were fabricated by coassembly of ESIONs, ligand A and ligand B (Scheme 2) ( FIG. 17 ). 
     Since ESIONs (about 3 nm) were much smaller than the hydrodynamic dimensions of the peptide block of the ligands (about 60 residues; length of about 20 nm), the catechol-anchored ligand A could wrap around the periphery of the ESIONs. These functionalized ESIONs can thus be considered polymer-metal analogues of conventional amphiphilic diblock copolymers since the functionalization permitted directed selfassembly of ESIONs into colloidal magneto-core shell structures (Scheme 3) ( FIG. 18 ). The hydrophobic core is then composed of the ESIONs and hydrophobic blocks of the tumor-sensing polymeric ligands. 
     Transmission electron microscopy (TEM) ( FIG. 5 a   ) revealed the particle size of PMNs is ˜60 nm They were well dispersed in water, and the hydrodynamic diameter measured by dynamic light scattering (DLS) was found to be 70±5 nm ( FIG. 5 a   ), which is appropriate for the enhanced permeability and retention (EPR) effect for passive tumor targeting. As a control, the present inventors fabricated pH-insensitive nanoparticle assemblies (denoted as InS-NPs) by assembling ESIONs and the platform ligand, which were similarly sized to PMNs ( FIG. 6 ). 
     New ligand design of the present invention enabled a two-stage pH activation leading to surface charge reversal in the tumor periphery for increased cell adsorption and permeation as well as endo/lysosomal pH dependent theranostic activity of PMNs. This pH-dependent structural transformation is schematically presented in  FIG. 5 b   . First, PMNs are slightly negatively charged at pH 7.4. The drop in pH within the tumor environment causes increased imidazole ionization which reverses the polarity of the complex causing it to swell ( FIG. 5 c   ), thereby enhancing their payload delivery and cell internalization due to electrostatic interactions with the vicinal anionic cells. Upon internalization, the particles further ionize as the endosomal pH decreases to 5.5-6.0. Here the hydrophobic interactions of the core of the PMNs weaken, and the ionized unimers repel each other leading to complete dissociation as confirmed by both DLS ( FIG. 5 c   ) and TEM ( FIG. 5 a   ). Acid-base titration ( FIG. 7 a   ) demonstrated a sharp drop in transmittance (% T) at the critical pH range of 5.5-6.0. When the pH was increased again, % T recovery was hysteretic, thereby demonstrating a reversible pH-dependent assembly/disassembly process. Consequently, the photoactivity, i.e. singlet oxygen generation (SOG) and fluorescence, of PMNs was quenched at pH 7.4 due to fluorescence resonance energy transfer. At pH &lt;6, the photoactivity was dramatically recovered upon disassembly, similar to that observed for the other self-assembled polymeric ligands ( FIGS. 7 b  and 7 c   ). Interestingly, PMNs showed a 2-fold lower critical aggregation concentration (˜0.01 mg/mL) than the self-assembled polymeric ligands possibly due to polymer chain entanglement indicating their improved colloidal stability. 
     The X-ray diffraction (XRD) pattern of PMNs was similar to that of ESIONs ( FIG. 8 ), and the PMNs show a weak magnetization because of the spin-canting effect, as illustrated in  FIGS. 9 a  and 9 b   . The pH-dependent structural transformation of PMNs in water is expected to affect proton relaxation. Considering the large number of high-spin Fe(III) ions with five unpaired electrons (S=5/2) on the surface of ESIONs, direct water coordination with Fe 3+  species is a major contributor to the longitudinal relaxivity (r1) of PMNs. The intensity variation of the T 1  MR phantom of PMNs at different pH values (same Fe concentration) was well matched with the corresponding fluorescent imaging results ( FIG. 7 d   ). PMNs showed an r1 of 3.30 mM −1 ·s −1  with a transverse relaxivity (r2) of 43.95 mM −1 ·s −1  at pH 7.4, and a concomitant r1-increase and r2-decrease was observed as the pH decreased from 7.4 to 5.5 ( FIG. 7 e   ). It is assumed as pH decreases, ligands become protonated and gain hydrophilicity. Consequently, both the number of coordinated water molecules and the duration of their coordination with Fe 3+  will increase. Moreover, as pH-induced disassembly occurs, separated ESIONs exhibit a lower r2 compared to initial PMNs. Although the efficiency of the contrast effect is evaluated in terms of relaxivity (r1), the r2/r1 ratio also plays an important role in positive T 1  imaging as an excessively high r2 may preclude their use as T 1  contrast agents. As the pH decreases, the r2/r1 ratio of PMNs significantly decreases along with r2. Finally, the PMNs have a specific r1 value of 3.87 mM −1 ·s −1  and a low r2/r1 ratio of 5.8 at pH 5.5, which results in a bright signal in T 1 -weighted imaging. Therefore, the positive T 1  MR contrast of PMNs was quenched at pH 7.4 but greatly recovered at pH 5.5, which indicates PMNs can be used for sensitive T 1  MR imaging of acidic tumor regions. In contrast, MR contrast of InS-NPs was not dependent on pH ( FIG. 10 ). To the best of the present inventors knowledge, this is the first demonstration of biocompatible iron oxide nanoparticles showing pH-sensitive T 1  contrast that can be used for tumor pH-sensitive T 1  MR imaging. Although Gd 3+ -based T 1  contrast agent can also be designed to respond to pH stimulus, they generally have short blood half-lives, preventing their accumulation in tumors for high-resolution tumor imaging. Furthermore, Gd 3+ -based contrast agents can be potentially toxic; severe side effects were observed in patients with renal failure that received gadolinium-containing contrast agents, such that the U.S. FDA recently released a warning regarding Gd 3+ -based MR contrast agents and nephrogenic system fibrosis (NSF). While such side effects are quite rare, occurring in less than 5% of patients, the present inventors believe iron oxide nanoparticles may provide a more biologically and metabolically compatible alternative. The use of biodegradable polymeric peptides for iron oxide-based PMN fabrication gives the final product a longer blood half-life and a greater biocompatibility for clinical translation. 
     Example 22. In Vitro and In Vivo MRI 
     MRI investigations were conducted using a 1.5 T MR scanner (Signa Excite; GE Healthcare) by using a Litz Coil (diameter, 100 mm; length, 85 mm; DOTY Scientific Inc., NC, USA). Spin-lattice and spin-spin relaxation times (T 1  and T 2 ) were measured using fast spin echo (FSE) sequence for different concentrations of PMNs in media with different pH values at room temperature. For T 1  measurements, the Field of View (FOV) was set to 75×75 mm, slice thickness (SL)=3 mm, Echo time (TE)=9.3 ms and Repetition Time (TR)=505.2, 525.0, 545.0, 565.0, 585.0, 605.0, 625.0, 645.0, 665.0, 685.0, 705.0, 730.0, 755.0, 805.0, 855.0, 905.0, 955.0, 1005.0, 1055.0, 1105.0 ms. For T 2  measurements, the following parameters were used: FOV=100 mm*100 mm, SL=3 mm, TR=4,000 ms, TE=10.9, 21.7, 43.5, 54.4, 65.2, 87.0, 119.6, 141.3, 163.1, 174.0 ms. The longitudinal (r1) and transverse (r2) relaxivities were calculated from r i =(1/T i −1/T i0 )/c, where c is the Fe concentration of PMNs in mM, T i  is the relaxation time at concentration c, T i0  is the relaxation time of water, and i=1 and 2 for T 1  and T 2 . The cellular MR images were acquired using FSE sequence (FOV=100 mm*100 mm, SL=3 mm) For T 1  measurement, TR=400 ms and TE=10 ms were used, whereas for T 2  measurement, TR=4,000 ms and TE=86.72 ms were used. For the MR diagnosis of tumor in vivo, PMNs were injected through the tail vein at a dose of 2 mg Fe/kg body weight. Mice were then placed in a 1.5 T MR scanner (Signa Excite, GE Healthcare), and FSE sequence was used with the followed parameters: FOV=90 mm×90 mm, SL=2 mm, Flip angle (FA)=90°, for T 1  MR imaging, TR=400 ms, TE=10 ms, and for T 2  MR imaging, TR=3,000 ms, TE=101 ms. The transverse section images were obtained and analyzed using Onis Dicom Viewer (DigitalCore, Tokyo, Japan). 
     PMNs showed higher cellular uptake at pH 6.8 than at pH 7.4 as evidenced by both fluorescence and flow cytometry results ( FIG. 11 a   ). In contrast, the cellular uptake of Ce6 and InS-NPs was not affected by changes in pH. For cell uptake, the present inventors followed the iron oxide nanoparticles by TEM ( FIG. 11 c   ) which shows uptake in endosomes. The present inventors also followed the Ce6 dye by confocal laser scanning microscopy (CLSM) ( FIG. 11 b   ) which shows the fluorescence of PMNs merges perfectly with that of Lysotracker Green. The existence of the Ce6 signal suggests PMNs indeed disassembled for fluorescence dequenching here in the endosome. Consequently, PMNs showed no cytotoxicity in the dark ( FIG. 11 d   ) but induced cell death much more efficiently than Ce6 under illumination ( FIG. 11 e   ) at pH 6.8. MR contrast and fluorescence of human colorectal carcinoma (HCT116) cells labeled with PMNs ( FIG. 11 f   ) further confirmed their dual-modal imaging capability. 
     The present inventors performed in vivo early stage tumor diagnosis with PMNs. Without conjugation of any tumor-targeting moiety and in contrast to InS-NP injection, PMN injection resulted in significant T 1  enhancement of ultrasmall HCT116 tumors of ˜3 mm in diameter ( FIGS. 12 a -12 c   ), thus confirming their successful tumor targeting and pH-dependent T 1  MR contrast effect. pharmacokinetic studies showed both PMNs (t 1/2,PMN =2.90 h) and InS-NPs (t 1/2,InS-NP =2.19 h) had long blood circulation times ( FIG. 12 d   ). However, notably, PMN accumulation in tumors was &gt;2-fold higher than that of InS-NPs ( FIG. 12 e   ). Moreover, PMNs also enabled high-resolution fluorescent imaging of tumors in mice ( FIG. 12 f   ). Macroscopic fluorescent imaging of excised organs demonstrated significant tumor accumulation of PMNs ( FIG. 13 ), and subcellular CLSM confirmed uptake of PMNs by tumor cells ( FIG. 12 g   ). These results indicate PMNs are promising candidates for highly efficient early diagnosis of cancer. 
     Example 23. In Vitro and In Vivo PDT 
     For in vitro PDT, HCT116 cells (5×10 4 /well) were incubated with the materials described above. Photosensitization experiments were then performed by irradiating the cells with a 670-nm laser source (Institute of Electronics) for 2 min. The power at the level of the cells was fixed at 5 mW/cm 2 . Cell viability was assessed using the MTT assay. Absorbance intensity was measured at 595 nm using a microplate reader (Bio-Tek, VT, USA). A parallel set of control cells was also used in which cells incubated with the respective materials were not exposed to laser irradiation. For in vivo PDT, HCT116 tumors were developed in the flanks of 6-week-old male BALB/c nude mice by subcutaneously implanting HCT116 cells in 100 μL of a 1:1 mixture of Matrigel (BD Biosciences, Franklin Lakes, N.J.) and serum-free medium. Heterogeneous CT26 tumors were developed in the flanks of 6-week-old male BALB/c nude mice by subcutaneously implanting a mixture of CT26 (1×10 5  cells), CT26/MDR (1×10 5  cells), and M2-10B4 cells (1×10 5  cells) in 100 μL of serum-free medium. Ten days after inoculating the tumor cells, PDT treatment was performed as follows: group 1, saline only; group 2, free Ce6 only; group 3, InS-NPs; and group 4, PMNs (equivalent to 2 mg/kg body of Ce6). NIR images were obtained using KODAK image station (Image Station 4000 MM; Kodak, New Haven, Conn.) at 2 h post-injection. Laser treatment was performed in groups 1 through 4 by irradiating the tumor region with a 670-nm laser (250 mW/cm 2 , for 20 min) at 12 h post-injection. The PDT treatment described above was repeated 5 days later. The tumor size was measured 3 times a week after the first PDT treatment by using a Vernier caliper, and tumor volumes were calculated from width (a) and length (b) measurements (V=a 2 ×b/2, where a≦b). All mice were killed when the tumor measured 1,000 mm 3 , at the first sign of suffering, or between 15 and 21 day post-transplantation. 
     In order to explore the therapeutic effect of PMNs, in vivo photodynamic therapy (PDT) was performed ( FIG. 14 ). PDT is a clinically approved, minimally invasive therapeutic procedure for cancer treatment that relies on photosensitizers to produce cytotoxic singlet oxygen generation (SOG) upon irradiation. However, currently available photosensitizers lack tumor selectivity which causes undesirable damage to normal tissues. In our PMN system, the activity of the photosensitizer can be self-quenched until it reaches the target tumor site where this suppression can be rapidly reversed by tumor pH stimulus, in particular intracellular pH stimulus, leading to a highly specific region of effect. HCT116 tumor-bearing mice injected with PMNs and then irradiated showed significant tumor regression relative to mice treated with InS-NPs or free Ce6 ( FIGS. 14 d  and 14 e   ). In particular, at 1 week post-injection, the tumors were almost completely destroyed with only scar tissue remaining. The enhanced antitumor effect was confirmed by hematoxylin and eosin staining (H&amp;E) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. These results clearly demonstrated that pH-targeted PDT using PMNs was successful in homogeneous HCT116 xenografts. 
     In order to be a better match to clinical cancer treatment, the present inventors further demonstrated the therapeutic effect of PMNs in tumors of more heterogeneous and drug-resistant nature. The present inventors hypothesized the pH-targeting approach of PMNs would not be influenced by tumor heterogeneity, and since PDT kills by a nonspecific mechanism, drug-resistant cells are equally as susceptible as their naive counterparts. To test this hypothesis, the present inventors developed highly heterogeneous and drug-resistant CT26 tumors in mice. The heterogeneous CT26 tumors overexpress P-glycoprotein (P-gp), which is involved in the active efflux of anti-cancer agents, and stromal cell-derived factor-1 (SDF-1), which induces angiogenesis (CD31) ( FIG. 14 c   ). Consequently, the CT26 tumors provided a physiologically relevant model of clinical tumors with a much faster growth rate than homogeneous HCT116 tumors. Notably, InS-NPs produced some minor tumor growth inhibition in both the homogeneous HCT116 and heterogeneous CT26 tumors ( FIGS. 14 f  and 14 g   ) compared to the untreated control. However, the heterogeneous model grows much more aggressively such that the positive effects observed for InS-NPs are still far too weak. In contrast, PMNs provided the same dramatic tumor destruction in both CT26-tumor-bearing mice and homogeneous HCT116-tumor-bearing mice ( FIGS. 14 f  and 14 g   ). In the PMN-treated group, many cells in the tumor tissue and microvasculature as well as fibroblasts showed considerable destruction, whereas no obvious damage was observed in groups treated with InS-NPs or Ce6 ( FIGS. 14 f  and 14 g   ). Therefore, pH targeting appears to play an important role in the improved anti-cancer therapeutic efficacy of PMNs. Consequently, for the first time, the present inventors successfully demonstrated treatment of drug-resistant heterogeneous tumor via pH-sensitive PDT, indicating PMNs can be applicable to various tumor treatments including highly drug-resistant heterogeneous tumors. Furthermore, no significant loss in body weight was observed throughout the PDT treatment ( FIG. 15 ), and a series of in vivo biocompatibility tests including a histopathology examination and serum chemistry test ( FIG. 16 ) demonstrated PMNs are highly biocompatible.