Fucoidan nanogels and methods of their use and manufacture

Abstract

Described herein are polymeric drug-carrying nanogels that are capable of targeting to P-selectin for the treatment of cancer and other diseases and conditions associated with P-selectin. Furthermore, in certain embodiments, the nanogels presented here offer a drug release mechanism based on acidic pH in the microenvironment of a tumor, thereby providing improved treatment targeting capability and allowing use of lower drug doses, thereby reducing toxicity.

Claims

1. A polymeric nanoparticle with affinity to P-selectin, the nanoparticle comprising a non-covalent assembly of: (i) a sulfated polymer species comprising free hydroxyl moieties and free sulfate moieties capable of targeting P-selectin; and (ii) a hydrophobic drug, wherein the nanoparticle has an average particle diameter of between about 20 and about 200 nm, and wherein the non-covalent assembly is a self-assembly of the sulfated polymer species around the hydrophobic drug.

2. The nanoparticle of claim 1, wherein the sulfated polymer species comprises one or more members selected from the group consisting of a sulfated polysaccharide, protein, and a fucoidan.

3. The nanoparticle of claim 1, wherein the sulfated polymer species comprises a fucoidan.

4. The nanoparticle of claim 1, wherein the hydrophobic drug comprises a member selected from the group consisting of paclitaxel, MEK162, docetaxel, Camptothecin, sorafenib, ispinesib, LY294002, Selumetinib, PD184352, 5-fluorouracil, Cyclophosphamide, Atorvastatin, Lovastatin, etoposide, dexamethasone, gemcitabine, Rapamycin (Sirolimus), and methotrexate.

5. The nanoparticle of claim 1, wherein the nanoparticle has an average particle diameter of from about 100 nm to about 200 nm.

6. The nanoparticle of claim 1, wherein the nanoparticle further comprises a fluorophore.

7. The nanoparticle of claim 6, wherein the fluorophore is a near infra-red dye.

8. The nanoparticle of claim 7, wherein the infra-red dye is IR783.

9. A method for manufacturing the nanoparticle of claim 1, the method comprising: contacting the hydrophobic drug to the sulfated polymer species to form a mixture; and agitating the mixture to form nanoparticles.

10. The method of claim 9, wherein agitating the mixture comprises sonicating the mixture.

11. The method of claim 9, wherein the hydrophobic drug and the sulfated polymer species are precipitated together.

12. The method of claim 9, wherein the hydrophobic drug and the sulfated polymer species are precipitated together via nano-precipitation.

13. The nanoparticle of claim 1, wherein the nanoparticle is negatively charged.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The foregoing and other objects, aspects, features, and advantages of the present disclosure will become more apparent and better understood by referring to the following description taken in conjunction with the accompanying drawings, in which:

(2) FIG. 1 is a schematic diagram illustrating the preparation of pH-sensitive fucoidan nanogels for the delivery of doxorubicin (FiDOX), according to an illustrative embodiment of the invention.

(3) FIG. 2A shows vials containing fucoidan-paclitaxel nanoparticles (FiPXL), according to an illustrative embodiment of the invention.

(4) FIG. 2B shows the chemical structure of paclitaxel (PX) and fucoidan (Fi) in the fucoidan-paclitaxel nanoparticles, according to an illustrative embodiment of the invention.

(5) FIG. 3A are plots of dynamic light scattering measurements of FiDOX nanogels, according to an illustrative embodiment of the invention.

(6) FIG. 3B are transmission electron microscope images of nanogels, according to an illustrative embodiment of the invention.

(7) FIG. 4 is a graph showing rate of release of doxorubicin from FiDOX nanogels over time, as a function of pH, according to an illustrative embodiment of the invention.

(8) FIG. 5A shows a plot of fluorescence intensity demonstrating in vitro activity of FiDOX nanogels, according to an illustrative embodiment of the invention.

(9) FIG. 5B shows a plot of an MTT cell viability assay, according to an illustrative embodiment of the invention.

(10) FIG. 6 shows bioluminescence images demonstrating anti-tumor efficacy of FiDOX nanogels, according to an illustrative embodiment of the invention.

(11) FIG. 7 is a plot of bioluminescence showing anti-tumor efficacy of FiDOX nanogels, according to an illustrative embodiment of the invention.

(12) FIG. 8 is a plot showing laboratory mouse survival curve data following injection of FiDOX nanogel, according to an illustrative embodiment of the invention.

(13) FIG. 9 is a schematic showing fucoidan-ispinesib nanogels (Fi-ISP) and analogous nanoparticles, according to an illustrative embodiment of the invention.

(14) FIGS. 10A and 10B are electron micrographs of fucoidan-ispenesib nanoparticles (Fi-ISP) and PGA-ispinesib nanoparticles, according to an illustrative embodiment of the invention.

(15) FIG. 11 is an electron micrograph of fucoidan-MEK162 nanoparticles, according to an illustrative embodiment of the invention.

(16) FIGS. 12A-12C illustrate P-selectin expression in human cancers.

(17) FIG. 12A illustrates human tissue microarrays (TMA) stained with P-selectin antibody (Lymphoma normal tissue is from the spleen; Lymphoma 1: non-Hodgkin B cell lymphoma (Lymph node); 2: peripheral T cell lymphoma (Lymph node); 3: brain metastases of non-Hodgkin B cell lymphoma; Lung cancer 1: lung squamous cell carcinoma; 2: small cell undifferentiated carcinoma; 3: metastatic lung adenocarcinoma; Breast cancer 1: Infiltrating ductal carcinoma; 2: advanced infiltrating ductal carcinoma; 3: lymph node metastases of infiltrating ductal carcinoma.)

(18) FIG. 12B illustrates a percentage of positively stained samples from the TMAs calculated with imaging software.

(19) FIG. 12C illustrates data from The Cancer Genome Atlas showing P-selectin gene alterations in various cancers

(20) FIGS. 12D-12E illustrate a preparation scheme of P-selectin targeted nanoparticles.

(21) FIG. 12D illustrates preparation schemes for fucoidan-encapsulated paclitaxel nanoparticles (FiPAX) via nanoprecipitation (top) and doxorubicin-encapsulated fucoidan nanoparticles (FiDOX) (bottom) via layer-by-layer assembly, and SEM images of FiPAX and FiDOX nanoparticles (right).

(22) FIG. 12E illustrates binding of IR783 dye loaded FiPAX to immobilized human recombinant P-selectin after 15 min of incubation. Fluorescence was measured with a fluorescent plate reader.

(23) FIGS. 13A-13E illustrate anti-tumor efficacy of FiPAX vs. DexPAX with and without radiation.

(24) FIGS. 14A-14E illustrate selective endothelial/tumor penetration assessments in vitro.

(25) FIG. 14A illustrates assay to test penetration of nanoparticles into an activated endothelial monlayer barrier and infiltration into spheroids composed of tumor cells from a small cell lung cancer patient upon activation with TNF-α.

(26) FIG. 14B illustrates fluorescence of FiPAX or DexPAX nanoparticles in the upper and lower chambers was measured with a fluorescence plate reader at 780 nm (excitation) and 815 nm (emission) after 1 h of incubation.

(27) FIG. 14C illustrates the endothelial monolayer component of the chamber was visualized to estimate nanoparticle internalization using a fluorescent microscope equipped with a NIR sensitive XM10 Olympus CCD camera, binding/internalization of FiPAX or control DexPAX nanoparticles to a bEnd.3 endothelial cell monolayer (CellMask Green membrane stain) upon activation with TNF-α.

(28) FIG. 14D illustrates fluorescence images of nanoparticle penetration into tumor spheres upon endothelial activation.

(29) FIG. 14E illustrates quantification of tumor sphere uptake from 6 images per condition using ImageJ.

(30) FIGS. 15A-15F illustrate targeting P-selectin positive and negative tumors in-vivo.

(31) FIG. 15A illustrates high expression of P-selectin in a PDX model of small cell lung cancer (top), and fluorescence efficiency from IR783 loaded FiPAX and DexPAX injected to tumor bearing mice and imaged with IVIS 24 h and 72 h after injection, n=4 (bottom).

(32) FIG. 15B illustrates tumor growth inhibition of a P-selectin expressing small cell lung cancer PDX after a single treatment on day 12, n=10.

(33) FIG. 15C illustrates radiation induced expression of P-selectin in mice with bilateral 3LL tumors treated with 6 Gy gamma radiation on the right flank tumor only.

(34) FIG. 15D illustrates a percentage of P-selectin positive blood vessels from entire CD31 stained blood vessels. Data is presented as the mean of 4 images per timepoint at 10×.

(35) FIG. 15E illustrates fluorescence efficiency from IR783 loaded FiPAX and DexPAX injected to 3LL tumor bearing mice with or without treatment of 6 Gy gamma radiation on the right flank tumor only.

(36) FIG. 15F illustrates tumor growth inhibition via single administration of nanoparticles after radiation treatment. The data is presented as mean±standard error.

(37) FIGS. 16A-16D illustrate FiDOX efficacy in lung metastasis, P-selectin expression, and Bio distribution of FiDOX.

(38) FIGS. 17A-17E illustrate the efficacy of P-selectin targeted nanoparticles in metastases.

(39) FIG. 17A illustrates representative images of P-selectin and vasculature (CD31) staining in a B16F10 melanoma experimental lung metastasis model 14 days after inoculation.

(40) FIG. 17B illustrates survival data from two experiments using the B16F10 metastasis model treated with a single injection on day 7 after inoculation.

(41) FIG. 17C illustrates survival data from two experiments using the B16F10 metastasis model treated with a single injection on day 7 after inoculation.

(42) FIG. 17D illustrates bioluminescence images acquired 7 days after a single administration of treatment with FiDOX, free doxorubicin (DOX), fucoidan vehicle (Fi), or PBS control.

(43) FIG. 17E illustrates median photon count of the 6 treatment groups measured by IVIS and quantified by LivingImage software.

(44) FIGS. 18A-18E illustrate FiMEK improved pERK inhibition and efficacy.

(45) FIGS. 19A-19D illustrate inhibition of MEK improved anti-tumor efficacy and induced apoptosis by P-selectin targeted nanoparticles in vitro and in vivo.

(46) FIG. 19A illustrates proliferation of and A549 cell lines measured after 4 days of treatment with MEK162 or FI-MEK as indicated (top), and biochemical analysis of A375 and A549 cell lines treated for 4 hours with MEK163 or FI-MEK (bottom).

(47) FIG. 19B illustrates tumor growth of xenograft derived from A375 and SW620 treated once with vehicle, MEK162, FI-MEK or a daily dose of MEK162 (n=6).

(48) FIG. 19C illustrates biochemical (western blot) quantification of pERK and Cleavage PARP on xenografts A375 tumors treated for 2 and 16 hours with MEK163 or FI-MEK.

(49) FIG. 19D illustrates immunohistochemistry of Clevage PARP on xenogfrat HCT116 tumors treated with MEK162 or MEK-IR.

(50) FIG. 20A shows the size distribution of FiDOX, DexDOX, FiPAX, and DexPAX nanoparticles.

(51) FIG. 20B shows the zeta potential of FiDOX, DexDOX, FiPAX, and DexPAX nanoparticles.

(52) FIG. 20C shows SEM images of FiPAX and FiDOX nanoparticles. Scale bar is 100 nm.

(53) FIG. 20D shows that the sizes of FiDOX and FiPAX stays constant over a 5 day period.

(54) FIG. 20E shows the release of DOX over time for pH 7.4 and pH 5.5.

(55) FIG. 20F shows release of PXL over time for pH 7.4 and pH 5.5.

(56) FIG. 21 shows proliferation of cell lines was measured after 4 days of treatment with MEK162 or FI-MEK as indicated. Open circle-MEK162, Open Square-FiMEK.

(57) FIG. 22 shows the drug release profile MEK162 drug from nanoparticles over time at different pH.

(58) FIG. 23A shows IHC staining of P-selectin expression in MEK162 sensitive HCT116 and SW620 xenografts.

(59) FIG. 23B shows whole body imaging of FiMEK nanoparticles in A375 and SW620 xenografts 24 h post administration.

(60) FIG. 23C shows percentage % of tumor size change as calculated from day 0.

(61) FIG. 23D shows growth inhibition of different regiments.

(62) FIG. 23E shows evaluation of apoptosis after single administration of MEK162 or FiMEK.

DETAILED DESCRIPTION

(63) It is contemplated that methods of the claimed invention encompass variations and adaptations developed using information from the embodiments described herein.

(64) Throughout the description, where compositions are described as having, including, or comprising specific components, or where methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

(65) It should be understood that the order of steps or order for performing certain action is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

(66) The mention herein of any publication, for example, in the Background section, is not an admission that the publication serves as prior art with respect to any of the claims presented herein. The Background section is presented for purposes of clarity and is not meant as a description of prior art with respect to any claim.

(67) Fucoidan is a sulfated polysaccharide that is found in various species of brown algae and brown seaweed. It can be obtained and purified from natural sources, or it may be synthesized. In general, fucoidan has an average molecular weight of from about 10,000 to about 30,000 (e.g., about 20,000), but other molecular weights may be found as well. Naturally-occurring fucoidan includes F-fucoidan, which has a high content of sulfated esters of fucose (e.g., no less than 95 wt. %), and U-fucoidan, which contains sulfates esters of fucose but is about 20% glucuronic acid. The fucoidan used in various embodiments described herein contains no less than 50 wt. %, no less than 60 wt. %, no less than 70 wt. %, no less than 80 wt. %, no less than 90 wt. %, or no less than 95 wt. % sulfate esters of fucose.

(68) FIG. 1 is a schematic diagram 100 illustrating the preparation of pH-sensitive fucoidan nanogels for the delivery of doxorubicin (FiDOX). The pH sensitivity is conferred by hydrozone linkages between doxorubicin and polyethylene glycol (PEG). The fucoidan and DOX-PEG-DOX constructs are assembled via a layer-by-layer approach. Fucoidan (Fi) at 102 is contacted with the DOX-PEG-DOX construct (DPD) at 104 in the presence of a phosphonobile salt (PBS), thereby forming hydrogel aggregates at 106. The resulting aggregates are sonicated to form FiDOX nanoparticles. In one example, the particles had average diameter of from about 150 nm to about 170 nm, with a zeta potential of −55 mV.

(69) In various embodiments, the average particle diameter of FiDOX, or other drug-containing fucoidan nanogel, is from about 20 nm to about 400 nm, or from about 100 nm to about 200 nm, or from about 150 nm to about 170 nm. The average particle diameter may be measured, for example, via dynamic light scattering (DLS) of a nanogel dispersed in a solvent, or can be measured via transmission electron micrograph (TEM). In some embodiments, the nanogel has a substantially monodisperse particle size (e.g., has polydispersity index, Mw/Mn of less than 20, more preferably less than 10, and still more preferably less than 5, less than 2, or less than 1.5, e.g., has polydispersity index in the range from 0 to 1, e.g., from 0.05 to 0.3). Nanoparticles similar to FiDOX can be synthesized to encapsulate the drug vincristine, or other cationic drugs, by replacing the DOX-PEG-DOX construct with another drug construct containing the desired drug.

(70) FIG. 2A shows vials containing fucoidan-paclitaxel nanoparticles (FiPXL). These were prepared using a self-assembly approach, without chemical conjugation. This can be performed to encapsulate other drugs as well, such as ispinesib, MEK162, and sorafenib, for example. FIG. 2B shows the chemical structure of paclitaxel (PX) and fucoidan (Fi) in the fucoidan-paclitaxel nanoparticles.

(71) FIG. 3A shows plots of dynamic light scattering measurements of FiDOX nanogels, showing the particle diameter characterization is stable over at least seven days. FIG. 3B shows transmission electron microscope images of the FiDOX nanogels at different concentrations and magnification.

(72) FIG. 4 is a graph showing the percentage of released doxorubicin from FiDOX nanogels over time, as a function of pH. Low pH allows faster release due to the breakage of hydrazone bonds.

(73) FIG. 5A shows a plot of fluorescence intensity demonstrating in vitro activity of FiDOX nanogels. The binding of FiDOX to immobilized P-selectin was estimated by measuring fluorescence intensity of bound particles. Soluble fucoidan was able to inhibit binding. A recombinant human P-selectin protein was immobilized on an ELISA plate. FiDOX particles were added to the wells for 15 min and then washed. The bound particles were detected with a fluorescence plate reader. Free fucoidan was used to inhibit the binding of the particles to the immobilized P-selectin on the surface. The particles did not bind to immobilized albumin (BSA).

(74) FIG. 5B shows a plot of an MTT cell viability assay. The plot shows that FiDOX was more cytotoxic to B16F10 cells compared to polyglutamic acid-based nanogels.

(75) FIG. 6 shows bioluminescence images at day 21 of testing, demonstrating anti-tumor efficacy of FiDOX nanogels. A luciferase-expressing B16F10 melanoma lung metastasis model was used. The cells were injected into the tail vein at day 0. The FiDOX particles and controls were injected at day 7. The progression of metastasis was monitored with bioluminescence imaging after injection of luciferin. The bioluminescence images show the luciferase-expressing B16F10 cancer cells after injection of D-luciferin, 21 days after inoculation and 14 days after a single treatment. The FiDOX nanoparticles at 30 mg/kg and above clearly show more effective treatment than the untreated specimens, as well as specimens administered free DOX drug (not in nanoparticle form), or fucoidan nanoparticles without the DOX drug (Fi NPs).

(76) FIG. 7 is a plot of bioluminescence from the same test, showing anti-tumor efficacy of FiDOX nanogels. Here in FIG. 7, the median number of photons/sec/cm.sup.2/steradian was measured at given time points to demonstrate decreased tumor burden in FiDOX treated mice.

(77) FIG. 8 is a plot showing laboratory mouse survival curve data in the B16F10 melanoma lung metastasis model treated with a single injection of FiDOX nanoparticles, injected on day 7. The results compared favorably to an injection of free doxorubicin (DOX), fucoidan alone (Fi), and the untreated control.

(78) FIG. 9 is a schematic showing fucoidan-ispinesib nanogels (Fi-ISP) and analogous nanoparticles made by combining ispinesib with fucoidan or Poly Glutamic Acid (PGA) or PGA-PEG. Nanoparticles were formed by non-covalent assembly. Dynamic light scattering (DLS) plots are shown at 906, 910, and 914. FIGS. 10A and 10B are electron micrographs of the fucoidan-ispenesib nanoparticles (Fi-ISP) and PGA-ispinesib nanoparticles. FIG. 11 is an electron micrograph of fucoidan-MEK162 nanoparticles.

(79) 1 mg of MEK162 in 0.1 ml of DMSO was added dropwise to 15 mg of Fucoidan in 0.5 ml of sodium bicarbonate. The mixture was immediately sonicated for 2 min with a probe sonicator (40%) under ice. The mixture was centrifuged at 20.00 g for 20 min and the pellet was re-suspended in 1 ml PBS containing 1 mg of Fucoidan and was again sonicated for 2 min under ice. The particles were characterized with DLS, TEM and zeta potential measurements. 155 nm particles were obtained with −50 mV surface zeta potential.

Experimental Examples

Preparation of DOX-PEG-DOX (DPD)

(80) 10 mg of Hydrazide-PEG-hydrazide, NH2NH-PEG-NHNH2, MW 3400 (from NANOCS) and 10 mg Doxorubicin were dissolved in 3 ml methanol containing 100 μL of glacial acetic acid. The mixture was stirred in the dark for 24 h and then slowly precipitated in cold acetone/ether (2:1), collected with centrifugation (15,000 g, 20 min) and dried with vacuum. The product, DOX-PEG-DOX (DPD) was purified with Sephadex G25 PD10 desalting column with water as eluent and then lyophilized.

(81) Preparation of FiDOX and DexDOX Nanoparticles:

(82) Fucoidan from Fucus vesiculosus (SIGMA) and DPD were both dissolved in double distilled water and were mixed together at a weight ratio of 1:1 and formed immediate gel aggregates. The aggregates were collected with centrifugation (15,000 g 10 min) and re-suspended in PBS containing excess of ×5 Fucoidan. The mixture was sonicated with a probe sonicator 40% intensity (sonics vibra-cell) for 10 sec until a clear dark red solution appeared containing nanoparticles. The particles were collected with centrifugation (30,000 g 30 min), re-suspended in PBS, and sonicated in a bath sonicator for 10 min. The particles were characterized with DLS, TEM, and zeta potential measurement, and 150 nm particles were obtained with −55 mV surface zeta potential measurements (FIGS. 20A-B). FIG. 20C shows SEM images of FiDOX nanoparticles. Scale bar is 100 nm. FIG. 20D shows that the sizes of FiDOX stay constant over a 5 day period. FIG. 20E shows the release of DOX over time for pH 7.4 and pH 5.5. FIG. 20E shows release of PXL over time for pH 7.4 and pH 5.5.

(83) Preparation of FiPAX and DexPAX Nanoparticles:

(84) Paclitaxel-encapsulated fucoidan/dextran sulfate nanoparticles (FiPAX and DexPAX) were synthesized using a nano-precipitation method. 0.1 ml of paclitaxel dissolved in DMSO (10 mg/ml), was added drop-wise (20 μL per 15 sec) to a 0.6 ml aqueous polysaccharide solution (15 mg/ml) containing IR783 (1 mg/ml) and 0.05 mM sodium bicarbonate. The solution was centrifuged twice (20,000 G 30 min) and re-suspended in 1 ml of sterile PBS. The suspension of nanoparticles was sonicated for 10 sec with a probe sonicator at 40% intensity (Sonics). The resulted nanoparticles had zeta potential of −52 mV and a size of 95 nm with a PDI of 0.12 (FIGS. 20A-B). By suspending the nanoparticles in lower volumes, it was possible to solubilize Paclitaxel (PXL) up to 16 mg/ml in saline solution, which is 2000 times better than free drug. The nanoparticles were lyophilized with a saline/sucrose 5% solution and reconstituted in water at this concentration. FIG. 20C shows SEM images of FiPAX nanoparticles. Scale bar is 100 nm. FIG. 20D shows that the sizes of FiPAX stay constant over a 5 day period. FIG. 20E shows the release of DOX over time for pH 7.4 and pH 5.5. FIG. 20E shows release of PXL over time for pH 7.4 and pH 5.5.

(85) Preparation of Fucoidan—Albumin Nanoparticles Containing Sorafenib:

(86) 1 mg of Sorafenib (LC labs) in DMSO was added to 4 mg of Human Serum Albumin (HSA, Sigma) in 0.3 ml of PBS (pH 4 acidified with HCl) to form a milky white mixture. 3 mg of Fucoidan in 0.3 ml water was added to the mixture. The mixture was bath sonicated for 2 min and 0.3 ml of sodium bicarbonate 100 mM was added until pH 8 was reached. The mixture was sonicated with a probe sonicator for 20 sec under ice and white clear solution containing nanoparticles. The solution was centrifuged at 30.00 g for 20 min and the pellet was re-suspended in PBS followed by bath sonication. 90 nm particles were obtained with −42 mV surface zeta potential (FIGS. 20A-B).

(87) Preparation of Fucoidan Nanoparticles Containing Paclitaxel:

(88) 1 mg of Paclitaxel in 0.1 ml of ethanol was added dropwise to 5 mg of Fucoidan in 0.5 ml of water. The mixture was immediately sonicated for 2 min with a probe sonicator (40%) under ice. The mixture was centrifuged at 20.00 g for 20 min and the pellet was re-suspended in 1 ml PBS containing 1 mg of Fucoidan and was again sonicated for 2 min under ice. The particles were characterized with DLS, TEM and zeta potential measurements (FIGS. 20A-B). 180 nm particles were obtained with −51 mV surface zeta potential.

(89) While the invention has been particularly shown and described with reference to specific preferred embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

(90) Preparation of Fucoidan-Albumin Nanoparticles Containing Paclitaxel and Near-IR Dye:

(91) Conjugation of Fucoidan to BSA Via Maillard Reaction:

(92) 150 μl of BSA (20 mg/ml) was mixed with 150 μl of Fucoidan solution (80 mg/ml), then 150 μl of 0.1 M sodium bicarbonate buffer, pH 8.0, was added. The mixture was frozen at −80° C., freeze-dried, and heated at 60° C. for 5 hr. After heating, samples were dissolved in 1 ml of water, and purified with Sephadex G25 PD10 column to remove salts and unbound sugar, then freeze dried.

(93) Preparation of Particles from Ficoidan Conjugated BSA:

(94) The Fucoidan BSA conjugate (Fi-BSA, 15 mg) was dissolved in 0.5 ml of water. 0.1 mg of IR783 (Sigma) in water was added to the solution. 1 mg of Paclitaxel in 0.1 ml of ethanol was added dropwise and the mixture was sonicated with a probe sonicator for 1 min. The mixture was centrifuged at 20.00 g for 20 min and the pellet was re-suspended in 1 ml PBS. 110 nm particles were obtained with −45 mV surface zeta potential.

(95) Binding of Nanoparticles to Immobilized P-Selectin:

(96) Human recombinant P- and E-selectin (50 ng in 50 μl) was added to high hydrophobicity 96 well elisa plate and incubated at 4° C. overnight. The wells were washed with PBS, incubated with BSA (3% 0.2 ml), and incubated with FiPAX or DexPAX in Hank's balanced salt solution (HBSS) for 15 min. The wells were gently washed three times with HBSS and the binding of nanoparticles was evaluated using scanning fluorescence intensity performed by TECAN T2000 (‘multiple reads per well’ mode, ex 780 nm, em 820 nm).

(97) Binding of Nanoparticles to P-Selectin Expressing Endothelial Cells:

(98) To induce P-selectin expression, monolayers of bEnd3 cells in 24 well plates were pre-incubated with TNF-α (50 ng/ml) for 20 min prior to the onset of experiments. Control cells were left untreated. The cells were then incubated with 20 μg/ml of nanoparticle for 45 min and another 15 min with CellMask Green (Life Technologies) for membrane staining and HOESCHT 66XX for nuclear staining. The cells were then washed twice with PBS. Images were acquired with an inverted Olympus XX fluorescent microscope, equipped with XM10IR Olympus camera with an IR range and EXCITE Xenon lamp. Similar exposure time and excitation intensity were applied throughout all experiments. Merged images were obtained via processing with ImageJ. Green—Cell membrane (ex 488 nm, em 525 nm), Blue—Nucleus (ex 350 nm, em 460 nm), Red—IR783 dye in particles (ex 780 nm, em 820 nm).

(99) Evaluation of Penetration Through Endothelial and Epithelial Barriers:

(100) A modified Transwell assay was used to test penetration of particles through a monolayer of endothelial cells expressing P-selectin.

(101) bEnd3 cells (5′ 10.sup.4 in 0.5 ml) were grown on Transwell inserts in 24 wells plate for 7 days. The medium was replaced every other day. The confluence of the monolayer was validated with imaging of membrane cell staining to validate the lack of gaps between cell junctions. Following activation by TNF-α as described above, the cells were incubated with 20 μg/ml of nanoparticles for 1 h and then samples from the upper chamber (50 μl) and fluorescent intensity was measured with a fluorescence plate reader (TECAN T2000) at ex 780 nm, em 820 nm. To visualize the particles in the endothelial cells on the insert component of the chamber, the cells were washed twice with PBS and then incubated in HBSS. Images were acquired and processed as described above.

(102) Cell Viability Assay:

(103) bEnd3 cells (5×10.sup.4) were seeded in a 96-well plate. Nanoparticles were added to cells that were pre-activated by TNF-α for 30 min, at equivalent drug concentration, and were incubated for 1 h at 37° C. Cells not activated with TNF-α were treated similarly. The drug solution was then removed and replaced with fresh medium, followed by 72 h of incubation at 37° C. Cell survival was assayed by discarding the medium and adding 100 μl of fresh medium and 25 μl of 5 mg/ml MTT solution in PBS to each well. After 90 minutes, the solution was removed and 200 μl of DMSO were 10 added. Cell viability was evaluated by measuring the absorbance of each well at 570 nm relative to control wells.

(104) Anti-Tumor Efficacy in Bilateral s.c Model of 3LL:

(105) Murine Lewis lung carcinoma (LLC) were maintained in Dulbecco's Modified Eagle Medium (DMEM) cell culture medium supplemented with 10% fetal bovine serum, 1 mM Na pyruvate, and 50 ug/ml penicillin and streptomycin. Tumor cells were subcutaneously implanted (1×10.sup.6 cells per injection) in both hind limbs of eightweek old hairless SKH-1 mice. The tumor models were used for biodistribution and tumor growth studies when the tumor size reached 0.5 cm in diameter.

(106) Irradiation of the tumors was conducted at 6 gy doses using X-ray irradiator.

(107) Near Infrared Imaging In Vivo:

(108) Four hours after irradiation, 200 μl (1 mg/ml) of the nanoparticles labeled with IR783 were injected via the tail vein. Biodistribution of the particles within the tumor-bearing mice was monitored with near infrared (NIR) imaging. NIR images were taken with an IVIS imaging system at various time points. Radiance (photons/sec/cm.sup.2) was measured within the tumor region (region of interest, ROI) using the program LivingImage 4.2 provided by Xenogen.

(109) Inhibition of Tumor Growth and Lung Metastasis of B16-F10 Melanoma:

(110) C57BL/6 mice were inoculated intravenously (i.v.) with 1×10.sup.5B16-F10 cells on day 0 and the tumor was allowed to establish until day 7. In one experiment, mice were divided randomly into 5 groups and injected i.v. with FiDOX, Fi, DexDOX.

(111) After treatment, mice were monitored up to 8 or 17 weeks, depending on the treatment received. At the end of the experiments, mice were sacrificed, their lungs were collected, and the number of surface-visible tumors was examined. The Kaplan-Meier method was used to evaluate survival.

(112) Establishment of Tumor Xenografts and Studies in Nude Mice:

(113) Six-week-old female athymic NU/NU nude mice were injected subcutaneously with 5×10.sup.5 of A375, SW620, LOVO, and HCT116 in 100 ml culture media/Matrigel at a 1:5 ratio. For cell-line-derived xenografts, animals were randomized at a tumor volume of 70 to 120 mm.sup.3 to four to six groups, with n=8-10 tumors per group. Animals were orally treated daily with MEK162 (10 mg/kg or 30 mg/kg in 0.5% carboxymethylcel-lulose sodium salt [CMC]; Sigma). Xenografts were measured with digital caliper, and tumor volumes were determined with the formula: (length×width.sup.2)×(π/6). Animals were euthanized using CO.sub.2 inhalation. Tumor volumes are plotted as means±SEM. Mice were housed in air-filtered laminar flow cabinets with a 12-hr light/dark cycle and food and water ad libitum.

(114) Immunohistochemistry (IHC):

(115) For xenograft samples, dissected tissues were fixed after (e.g, immediately after) removal in a 10% buffered formalin solution for a maximum of 24 h at room temperature before being dehydrated and paraffin embedded under vacuum. The tissue sections were deparaffinized with EZPrep buffer, antigen retrieval was performed with CC1 buffer, and sections were blocked for 30 minutes with Background Buster solution (Innovex). Human P-Selectin/CD62P Monoclonal Antibody (Catalog #BBA30) at 5-15 μg/mL overnight at 4° C. Other antibodies (CD31, P-selectin IFC, Tunel and Cle-PARP) were applied and sections were incubated for 5 hr, followed by a 60 minute incubation with biotinylated goat anti-rabbit IgG (Vector labs, cat#PK6101) at a 1:200 dilution.

(116) As described herein, it has been identified that human tumors (e.g., lymphomas) express P-selectin primarily on cancer cells and to a lesser extent in the vasculature. Because of the augmented expression on certain tumor cells and vasculature, P-selectin was tested on targeted nanoparticles in a murine model that express P-selectin in both cancer and endothelial cells, models that only express endothelial P-selectin, and models that do not express P-selectin but it can be induced by radiation. For each of the models, appropriate drugs were chosen to achieve high response to a single injection, which demonstrated the platform capabilities of Fi-based nanoparticles.

(117) There was a significant increase in fucodian particle accumulation in P-selectin expressing tumors on cancer cells and endothelial cells (PDX and irradiated 3LL) in tumor bearing mice. An active mechanism of delivery of the chemotherapeutic agents loaded fucoidan nanoparticles (FiDOX and FiPAX) in P-selectin positive aggressive lung metastases and PDX models was not seen in the control nanoparticles with similar charge and size (DexDOX and DexPax. To further characterize the pharmacodynamics of fucoidan based particles, the activities of a reversible kinase inhibitor were investigated. The use of a reversible MEK inhibitor encapsulated in fucoidan nanoparticles allowed evaluation of kinase inhibition in cancer cells and correlation with drug delivery to cancer cells. Comparison of a clinically relevant regimen of daily administration of MEK162 to a single or weekly dose of the nanoparticle formulation was performed. A single or a weekly administration of a reversible inhibitor such as MEK162 encapsulated in a nanoparticle was similar to or more effective as a daily administration. This demonstrates the effectiveness of the delivery system to reach not just endothelial cells but also cancer cells. The reduction of a chronic and systemic inhibition of the pathway and the increase in local tumor concentrations for prolonged periods of time using Fi nanoparticles will be more efficacious and better tolerated.

(118) Because the overexpression of P-selectin on endothelial cells and cancer cells varies substantially from patient to patient, radiation was examined as a way to induce P-selectin locally. In tumors without P-selectin expression, it was demonstrated that radiation increases endothelial P-selectin levels as well as particle accumulation and anti-tumor efficacy. The ability to ‘turn on’ expression of and translocation of P-selectin using radiation has a unique advantage since it could render virtually any tumor vulnerable to P-selectin targeted systems. Also, unexpectedly, non-irradiated tumors experienced a significant therapeutic benefit by a mechanism which may be akin to the abscopal effect.

(119) P-selectin was investigated as a target for localized drug delivery to tumor sites, including metastases. It was found that many human tumors surprisingly express P-selectin spontaneously within their stroma, tumor cells, and tumor vasculature. A nanoparticle carrier was synthesized for chemotherapeutic and targeted therapies using the algae-derived polysaccharide, fucoidan, which exhibits nanomolar affinity for P-selectin. It was found that the targeting of activated endothelium improved the penetration of fucoidan-based nanoparticles through endothelial barriers, leading to a therapeutic advantage in P-selectin-expressing tumors and metastases. The encapsulation of both chemotherapeutic drugs and a reversible MEK inhibitor conferred a therapeutic benefit in P-selectin-expressing tumors, suggesting improved delivery to tumor tissue. On exposing tumors to ionizing radiation, which induced expression of P-selectin, a significant increase in nanoparticle localization and anti-tumor efficacy in tumors that do not spontaneously express the target was observed.

(120) Expression of P-Selectin in Human Cancers:

(121) In order to determine the prevalence of P-selectin expression in cancer tissues, ˜400 clinical samples were assessed via immunohistochemistry. (Table S1). As shown in FIGS. 12A and 12B, it was found that P-selectin is highly expressed within multiple types of tumors and their metastases, including human lung (19%), ovarian (68%), lymphoma (78%) and breast (49%). Abundant expression of P-selectin was found in the stroma and vasculature surrounding the tumor cells. However in a subset of cancers, expression of P-selectin on tumor cells was observed. Moreover, significant genomic alterations to the P-selectin gene (SELP) were noted in The Cancer Genome Atlas (TCGA) (FIG. 12C). It was found that SELP is amplified in many cancers including breast (27.5%), liver (15%), bladder urothelial carcinoma (13.4%), and lung adenocarcinoma (12.2%). Moreover, the expression of SELP is associated with poor prognosis in squamous cell carcinoma of the lung and renal cell carcinoma. (FIG. 12C). The abundant expression of P-selectin in cancer prompted the development a P-selectin-targeted vehicle for selective drug delivery.

(122) P-Selectin Mediated Transport of Nanoparticles:

(123) To design a P-selectin targeted drug delivery system, fucoidan (Fi)-based nanoparticles were prepared to encapsulate three different drug classes with dose-limiting toxicities. Fucoidan-encapsulated paclitaxel (PAX) nanoparticles (FiPAX) were synthesized by co-encapsulating paclitaxel, and a near infra-red dye (IR783) to facilitate imaging, via nano-precipitation as described above in Preparation of FiPAX and DexPAX nanoparticles (FIG. 12D). A reversible MEK inhibitor, MEK162 was encapsulated in fucodian nanoparticles (FiMEK) in the same manner that FiPAX was prepared. Fucoidan-encapsulated doxorubicin (DOX) nanoparticles (FiDOX) were synthesized via layer-by-layer assembly of a cationic doxorubicin-polymer conjugate via pH sensitive hydrazone bond (DOX-PEG-DOX, DPD) and the anionic fucoidan (FIG. 12E). The DPD conjugate was synthesized via pH-cleavable hydrazine linkages to promote release of the drug in the acidic tumor microenvironment or lysosomes. The FiDOX, FiPAX and FiMEK nanoparticles measured 150±8.1, 105±4.2 and 85±3.6 nm in diameter respectively, and exhibited approximately −55 mV surface charge (zeta potential). Microscopy showed relatively uniform spherical morphology. As shown in Table 1 below and in FIGS. 13A-E, the particles exhibited good serum stability and reconstituted after lyphilization.

(124) TABLE-US-00001 TABLE 1 Parameters (units) Control (NT) FiDox (24 Hrs) FiPax (24 Hrs) WBCs (K/μL)  6.90 ± 0.91  4.71 ± 0.28 5.21 ± 0.70 NE (K/μL)  3.71 ± 3.23  1.46 ± 0.12 1.61 ± 0.48 LY (K/μL)  3.03 ± 2.41  3.20 ± 0.29 3.51 ± 0.26 MO (K/μL)  0.15 ± 0.05  0.06 ± 0.03 0.07 ± 0.02 EO (K/μL)  0.03 ± 0.02  0.01 ± 0.01 0.01 ± 0.01 BA (K/μL)  0.00 ± 0.00  0.00 ± 0.00 0.00 ± 0.00 RBC (M/μL) 10.30 ± 0.69 10.55 ± 0.60 10.55 ± 0.54  Hb (g/dL) 13.30 ± 0.99 13.87 ± 0.50 13.70 ± 0.52  HCT (%) 45.05 ± 2.05 45.73 ± 1.96 45.30 ± 2.78  MCV (fL) 43.75 ± 0.92 43.40 ± 1.14 42.90 ± 0.95  MCH (pg) 12.90 ± 0.14 13.20 ± 0.61 13.00 ± 0.40  MCHC (g/dL) 29.50 ± 0.85 30.40 ± 0.69 30.30 ± 0.89  PLT (K/μL) 1082.00 ± 203.65 753.33 ± 50.08 890.33 ± 125.92

(125) To assess the selectivity of nanoparticle targeting to P-selectin, an untargeted control drug-loaded nanoparticle lacking the fucoidan component was synthesized. Dextran sulfate-encapsulated paclitaxel (DexPAX) nanoparticles were assembled with the same methods as used above. The binding of FiPAX and DexPAX was compared to immobilized human recombinant P-selectin, E-selectin, and BSA, thereby confirming the selective binding to P-selectin in a dose dependent manner (FIG. 12E: P<0.05).

(126) It was investigated whether a fucoidan-based nanoparticle would bind to activated endothelium and translocate the endothelial barrier. The ability of fucoidan nanoparticles to penetrate through endothelium and into tumor tissue was assessed using a modified Transwell assay. Murine brain endothelial (bEnd.3) cells were grown on the top chamber's membrane, and P-selectin expressing tumor spheroids were grown in the bottom chamber (FIG. 14A). The penetration of the nanoparticles through the bEnd.3 monolayer upon activation by TNF-α was measured. The quantity of FiPAX nanoparticles recovered from the bottom chamber increased significantly by ˜30% (FIG. 14B) in the presence of TNF-α, while DexPAX increased by 15%, suggesting that endothelial activation enhanced the translocation of the FiPAX nanoparticles. The FiPAX nanoparticles were taken up by the endothelial cells only upon activating with TNF-α, and the cells did not take up the control DexPAX nanoparticle in either case (FIG. 14C). Penetration of the nanoparticles into tumor spheres via fluorescence microscopy was quantified. As shown in FIGS. 2D to 2E, a 3-fold increase in the FiPAX-encapsulated dye fluorescence in the tumor spheres upon activation with TNF-α, as well as greater penetration into the spheres, compared to the DexPAX nanoparticles (FIGS. 14D-14E). These observations suggest that endothelial activation mediates increased transport of P-selectin-targeted nanoparticles across an endothelial barrier and into solid tumor tissue compared to untargeted particles. These findings support that particle extravasation and tumor penetration to P-selectin expressing tumors in vivo is possible.

(127) Anti-Tumor Efficacy Mediated by P-Selectin:

(128) To determine the net efficacy of P-selectin targeting in vivo, a patient-derived xenograft (PDX) model of SCLC which expresses P-selectin was used (FIG. 15A). This PDX expressed P-selectin both in tumor endothelium and cancer cells (FIG. 13A: LX36). When tumors reached 70 mm.sup.3, mice were randomized into 4 arms: PBS, FiPAX, DexPAX and paclitaxel (PAX). Upon 24 h and 72 h after injection of nanoparticles, the mice were imaged to compare particle localization. The average fluorescence intensity was 2.5 times higher than that of DexPAX after 24 h, and the signal difference increased to 4.1 times at 72 h (FIG. 15A, FIG. 16B). Upon administration of a single injection of each treatment, FiPAX nanoparticles significantly inhibited tumor progression as compared to free paclitaxel or untargeted DexPAX nanoparticles (FIG. 15B).

(129) To investigate the radiation-induced expression of P-selectin in a model that does not spontaneously express the target, nude mice were inoculated in both flanks with Lewis lung carcinoma (3LL) cells. The resulting tumor did not endogenously express P-selectin, as observed by tissue staining (FIG. 15C). The right flank tumor of each mouse was irradiated with 6 Gy, while the left tumors were left un-irradiated. It was observed that the expression of P-selectin in the irradiated tumor was apparent by 4 hours and increased substantially by 24 hours (FIG. 15C). Notably, P-selectin expression was found in the non-irradiated tumors of the irradiated mice after a 24 hour delay (FIG. 15C), as well as an increase in soluble P-selectin (sP-selectin) in the blood of the irradiated mice (FIGS. 16A-16D).

(130) It was investigated whether radiation could selectively guide P-selectin-targeted drug carrier nanoparticles to a tumor site to result in a net therapeutic benefit. The 3LL bilateral tumor model was irradiated with 6 Gy on the right tumor before injecting the mice i.v. with nanoparticles 4 hours later. To distinguish the effects of radiation-induced P-selectin targeting from an EPR effect or non-induced P-selectin, untargeted DexPAX nanoparticles and non-irradiated control mice were included. At 24 hours after treatment, the fluorescence signal from FiPAX nanoparticles were 3.8 times higher in the irradiated tumors over non-irradiated tumors, while there was no difference in the DexPAX-treated mice (FIG. 15E, 16D). Growth was halted in tumors receiving both radiation and FiPAX nanoparticles, resulting in their complete tumor disappearance (FIG. 15F). Notably, in mice treated with FiPAX nanoparticles and radiation, significant inhibition was observed in the non-irradiated tumors, suggesting an abscopal-like effect on anti-tumor efficacy mediated by the nanoparticle. To corroborate the in vivo observations, FiPAX binding to radiation-induced P-selectin expression in vitro were evaluated. In bEnd.3 endothelial cells, radiation-mediated P-selectin expression was observed in a dose-dependent manner. The irradiated cells took up FiPAX nanoparticles, while little uptake of DexPAX nanoparticles was measured (FIGS. 16A and 16B: P<0.05).

(131) Anti-Tumor Efficacy in Endogenous P-Selectin Expressing Metastases:

(132) The anti-tumor efficacy of P-selectin-targeted drug carrier nanoparticles was assessed against an aggressive experimental metastasis model. The i.v. injection of 10′ B16F10 melanoma cells results in lung metastases which exhibit P-selectin expression in the associated vasculature (FIG. 17A-B). Three different doses of FiDOX (fucoidan-encapsulated doxorubicin) nanoparticles were then administered to identify a therapeutic window. The mice were divided into 6 groups of 5 mice and treated with a single dose of either free doxorubicin at 6 mg/kg or 8 mg/kg, close to the maximum tolerated dose, fucoidan (30 mg/kg), as a vehicle control, and three concentrations of Fi DOX (1 mg/kg, 5 mg/kg and 30 mg/kg). The treatment with Fi DOX nanoparticles at all three concentrations resulted in decreased tumor burden and prolonged survival upon a single injection, whereas an equivalent amount of free doxorubicin at its maximum tolerated dose, did not have a significant effect (FIG. 17C). Fucoidan alone also showed no survival benefit. After 7 days post-treatment, tumor bioluminescence shows a clear reduction in median photon count in the medium and the high dose groups (FIG. 17D, 17E). Signs of toxicity as measured by weight loss or complete blood count were not observed (FIGS. 18A-18E). The anti-tumor efficacy of FiDOX nanoparticles was also compared to the untargeted DexDOX nanoparticle control and untargeted drug-polymer conjugate, DOX-PEG-DOX at equivalent doxorubicin doses of 8 mg/kg. The mean survival of the FiDOX group was significantly higher at 68.8 days with 400 cured mice compared to DexDOX at 40.2 days with no cures, DOX-PEG-DOX at 39.2 days, and untreated 32.4 at days (FIG. 17B, p=0.005).

(133) P-Selecting Targeting of Mechanistically Targeted Drugs:

(134) The Ras-ERK pathway is frequently hyperactive in substantial types of cancers including melanoma, colorectal, and lung cancers, and therefores MEK/ERK reversible inhibitors have been tested in large number of clinical trials in RAS- and BRAF-mutated cancers. Blocking this pathway using systemic MEK/ERK inhibitors is, however, dose-limiting with only temporal target inhibition. At high dosage, these treatments cause toxicity in patients such as severe rash and chronic serous retinoscopy (CSR).

(135) It is described herein how P-selectin-targeted delivery improved the efficacy of reversible kinase inhibitors which are specific to cancer cells. For example, the delivery of MEK inhibitor to the tumor microenvironment using P-selectin targeted nanoparticles increased the concentration of drug in the tumor itself, therefore prolonging the duration of inhibition and reduce systemic toxicity.

(136) To this end, MEK162 was co-encapsulated with IR783 within fucoidan-based nano-particles (FiMEK) in the same manner that FiPAX was prepared. In vitro, the release of the MEK162 by the nano-particle was sustained with maximum of 85% reached in 24 hours and accelerated by acidic pH (FIG. 23A). Data shows that free MEK162 and MEK162 loaded fucoidan nanoparticles (FI-MEK) had similar biochemical and anti-tumor activity against BRAF mutated melanoma (A375), and NRAS mutated lung (A549) and KRAS mutated colorectal (HCT116 and SW620) cancer cells in vitro (FIG. 19A, FIG. 21).

(137) In tumor bearing mice, a single administration of FIMEK induced significant tumor growth inhibition compared to no effect of oral treatment. A375 and SW620 tumor bearing mice were treated with a weekly dose of MEK162, FiMEK and a daily dose of free MEK162. It was observed that a weekly dose of FiMEK was more effective than a weekly dose of free MEK, and had comparable efficacy with a daily administration. This result was validated in two other models of LOVO and HCT116 xenografts (FIGS. 23A-23E).

(138) To further understand the enhanced efficacy of FiMEK compared to oral MEK162, the pharmacodynamics were assessed by measuring the levels of pERK and cleavage of PARP on tumors treated with MEK162 or FiMEK at 2 h and 16 h after administration (FIG. 19C). The data shows a similar inhibition of pERK after 2 hours of treatment. However, significant prolong of pERK inhibition was observed in mice treated with FIMEK when compared to mice treated with oral MEK162. An association between prolong inhibition of ERK and induction of apoptosis was observed, indicating the importance of the duration of pathway inhibition. Immunohistochemistry of Clevage PARP on xenogfrat HCT116 tumors treated with MEK162 or MEK-IR was assessed to confirm the death of tumor cells (FIG. 19D, 19E). FIG. 22 shows the drug release profile MEK162 from nanoparticles over time at different pH.