Patent Application
20110275686
1. Field of the Invention
The present invention concerns the preparation of nanoparticle formulations containing hydrophobic photosensitizers and their use in photodynamic therapy, particularly for photodynamic tumor therapy, using intravenous administration.
2. Information Disclosure Statement
Photodynamic therapy (PDT) is one of the most promising new techniques now being explored for use in a variety of medical applications and particularly is a well-recognized treatment for the destruction of tumors. Photodynamic therapy uses light and a photosensitizer (a dye) to achieve its desired medical effect. A large number of naturally occurring and synthetic dyes have been evaluated as potential photosensitizers for photodynamic therapy. Perhaps the most widely studied class of photosensitizers is the tetrapyrrolic macrocyclic compounds. Among them, especially porphyrins and chlorins have been tested for their PDT efficacy.
Porphyrins are macrocyclic compounds with bridges of one carbon atom joining pyrroles to form a characteristic tetrapyrrole ring structure. There are many different classes of porphyrin derivatives including chlorins containing one dihydro-pyrrole unit and bacteriochlorins containing two dihydro-pyrrole units. Both mentioned porphyrin derivatives possessing potential for PDT can either be derived from natural sources or from total synthesis.
Compared to porphyrins, chlorins have the advantage that they possess a more favorable absorption spectrum, i.e. they have a more intense absorption in the red and near-infrared region of the electromagnetic spectrum. As light of longer wavelength penetrates deeper into the tissue it is thus possible to treat e.g. more expanded tumors, when PDT is employed for tumor therapy.
Nevertheless, the use of PDT for the treatment of various types of disease has been limited due to the inherent features of photosensitizers (PS). These include their high cost, extended retention in the host organism, substantial skin phototoxicity, low solubility in physiological solutions (which also reduces their usefulness for intravascular administration as it can provoke thromboembolic accidents), and low targeting effectiveness. These disadvantages, particularly of PS in the prior art, had led to the administration of very high doses of a photosensitizer, which dramatically increase the possibility of accumulation of the photosensitizer in non-damaged tissues and the accompanying risk of affecting non-damaged sites upon irradiation.
Efforts to reduce cost and to decrease background toxicity have been underway but are unrelated to the developments of the present invention. Work to improve solubility in physiological solutions, effects of skin phototoxicity, retention in host organism and to a lesser extent targeting effectiveness are the areas where the present invention provides new and non-obvious improvements on the use of PDT to treat various neoplasia, hyperplasia and related diseases.
Most substances successfully employed for photodynamic tumor therapy are lipophilic substances, which due to their inherent low solubility in water need to be formulated in a proper way. Therefore, there is a great need for new formulations of tetrapyrrole-based photosensitizers to enhance their uptake in the body and their bioavailability.
Nanoparticles are intensively investigated as carriers for lipophilic drug substances. In fact, a nanoparticle formulation of the anti-cancer drug Paclitaxel based on human serum albumin (HSA) has been approved recently by regulatory authorities in Europe and the USA.
Nanoparticles in general are solid colloidal particles, typically, ranging in size from 10 nm to 1000 nm. They consist of macromolecular materials in which the active ingredient is dissolved, entrapped or encapsulated, and/or to which the active principle is absorbed or attached. Many different sorts of nanoparticle material have been investigated, i.e. quantum dots, silica-based nanoparticles, photonic crystals, liposomes, nanoparticles based on different polymers of natural and synthetic origin, and metal-based nanoparticles.
Nanoparticles in combination with photosensitizers have been investigated i.e. for many applications including imaging approaches, such as the nanoparticles, disclosed in Patent Publication N° US 2007/0148074A1 by Sadoqi et al., comprising biodegradable polymer materials entrapping near-infrared dyes for using them in bio-imaging. Additionally, other nanoparticle systems combining fluorescence imaging and magnetic resonance imaging, especially in combination with metal (iron) based nanoparticles are known in the art (see Mulder et. al, Nanomed., 2007, 2, 307-324; Kim et. al, Nanotechnol., 2002, 13, 610-614; Primo et al, J. Magnetism Magn. Mater., 2007, 311, 354-357) but such developments are unrelated to the present invention. Also, other nanoparticle formulations based on liposomes, quantum dots, inorganic materials (including metals) which are known in the art do not interfere with the present invention.
Most interesting as carrier systems for photosensitizers are nanoparticles that consist of biocompatible materials. Such carrier systems could significantly improve the treatment regimen of photodynamic therapy. A carrier system with such known high biocompatibility is e.g. poly(DL-lactic-co-glycolic acid) (PLGA). PLGA material has successfully been formulated as nanoparticles.
There are a few examples of PLGA-based nanoparticles as carriers for photosensitizers known in the art (see Gomes et al., Photomed. Laser Surg., 2007, 25, 428-435; Ricci-Junior et al., J. Microencapsul., 2006, 23, 523-538; Ricci-Junior et al., Int. J. Pharm., 2006, 310, 187-195; Saxena et al., Int. J. Pharm., 2006, 308, 200-204; McCarthy et al., Abstracts of Papers, 229th ACS Meeting, 2005; Vargas et al., Int. J. Pharm., 2004, 286, 131-145; Konan et al., Ear. J. Pharm. Sci., 2003, 18, 241-249; Konan et al., Ear. J. Pharm. Biopharm., 2003, 55, 115-124; Vargas et al., Ear. J. Pharm. Biopharm., 2008, 69, 43-53; Pegaz et al., J. Photochem. Photobiol. B: Biology, 2005, 80, 19-27).
Nevertheless, some of the known art mentioned above concentrates on other types of photosensitizers such as the invention disclosed in Patent Application No WO97010811A1 comprising the photosensitizers Zinc(II) phthalocyanine and indocyanine green which are unrelated to the present invention.
In other cases, such as in Patent Publication No WO03097096A1 and patent, U.S. Pat. No. 7,455,858 B2 by Allemann et al., the PLGA-based nanoparticles used as carriers for photosensitizers, are intended for a rapid release of the drug, preferably within about 60 seconds, after the nanoparticles are introduced into an environment containing serum proteins and, therefore, are not well suited for a drug transport to the target cells and tissues. There is a lack of targeting effectiveness of the PLGA-based nanoparticles as the photosensitizer is released within seconds after being introduced into an environment containing serum proteins. Furthermore, for the preparation of small sized and monodisperse PLA- or PLGA-based nanoparticles known in art high concentrations of polyvinyl alcohol (PVA) stabilizer in the range of about 5-20% in the aqueous phase are employed.
The application of a nanoparticle formulation for parenteral administration in clinical practice requires that the sterility of the formulation according to pharmacopoeial specifications can be assured. The problem of sterility of nanoparticle photosensitizer formulations involving PLGA is challenging because of the lability of the nanoparticle matrix material as well as the lability of the photosensitizer. Conventional methods of sterilization (autoclaving, use of ethylene oxide, gamma-irradiation) are incompatible with these photosensitizer formulations (see Athanasiou et. al, Biomaterials, 1996, 17, 93-102; Volland et. al, J. Contr. Rel., 1994, 31, 293-305). An alternative is the sterile filtration through membrane filters of a defined size for such chemically and thermally sensitive materials. Pore size for sterile filtration is usually 0.22 μm whereas nanoparticles of the present invention are in the size range between 100 and 500 nm. Therefore, sterile filtration has its drawbacks and is not generally compatible with the nanoparticles that are subject of the present invention.
Also, for a clinical application it is highly desirable that the formulation can be freeze dried and later be reconstituted in an aqueous medium. In particular, it is difficult to develop sterile nanoparticle formulations and nanoparticle formulations suitable for freeze drying in the case of photosensitizers of the present invention which are of the chlorin or bacteriochlorin type (i.e. tetrapyrroles carrying one or two dihydro-pyrrole units), because such systems are especially sensitive to oxidation and photo-chemical modifications induced by the handling conditions that are often used for nanoparticle preparation (see Hongying et al., Dyes Pigm., 1999, 43, 109-117; Hadjur et al., J. Photochem. Photobiol. B: Biology, 1998, 45, 170-178; Bonnett et al., J. Chem. Soc. Perkin Trans. 2, 1999, 325-328). These photosensitizers of the chlorin or bacteriochlorin type which possess one or two dihydro-pyrrole units, respectively, differ significantly in their chemical and physical behaviour from the corresponding porphyrins (see Bonnett et al., J. Chem. Soc. Perkin Trans. 2, 1999, 325-328; Bonnett et al., J. Porphyrins Phthalocyanines, 2001, 5, 652-661). The second point, that the problem of sterility and freeze-drying has up to now been addressed only for chemically more tetrapyrrole-based photosensitizers, holds especially for the green porphyrins described by Allemann et al. or for the photosensitizers investigated by Konan et al.
The PLGA-based nanoparticles used as carriers for photosensitizers known in the art either do not address such problems as sterility and freeze-drying or if so, the investigated photosensitizers are less problematic in this respect because of their more stable chemical structure.
This is the case of Patent Publication No WO 2006/133271 A2 which discloses Photosensitizer Nanoparticle Aptamer Conjugates comprising a photosensitizer that forms the central core of the nanoparticle, a biodegradable polymer shell and a targeting aptamer (e.g. ErbB3 receptor-specific aptamer) but does not address the problem of sterility of the nanoparticle photosensitizer formulations nor the freeze-drying process required to obtain a stable nanoparticle photosensitizer formulation.
In spite of the already mentioned drawbacks, present invention provides PLGA-based nanoparticle formulations and methods of preparation for photosensitizers suitable for parenteral application that can be prepared for such sensitive compounds as chlorins and bacteriochlorins.
There remains these problems in the art for which the present invention addresses and provides solutions.
It is an objective of the present invention to provide nanoparticle formulations for hydrophobic photosensitizers used for photodynamic therapy based on biocompatible PLGA material.
It is another objective of the present invention to provide nanoparticle formulations for hydrophobic photosensitizers of the tetrapyrrole type, namely chlorins and bacteriochlorins, based on poly(DL-lactic-co-glycolic acid) (PLGA) and a stabilizing agent, preferably selected from the group consisting of poly(vinyl alcohol), polysorbate, poloxamer, and human serum albumin and the like.
It is yet another objective of the present invention to provide nanoparticle formulations for hydrophobic photosensitizers which enable a high variation of photosensitizer loading efficiency (2 to 320 μg photosensitizer per mg nanoparticles) to the particle system giving the opportunity of a high variability in drug pharmacokinetics.
It is a further objective of the present invention to provide nanoparticle formulations for hydrophobic photosensitizers which enable an effective drug transport to target cells and tissues combined with a drug release after cellular accumulation
It is yet a further objective of the present invention to provide methods for the production of sterile PLGA-based photosensitizer-loaded nanoparticles of a mean particle size less than 500 nm, with the photosensitizers being chlorins or bacteriochlorins. The nanoparticles of the present invention are stable enough to allow freeze drying and reconstitution in an aqueous medium.
It is yet another object of the present invention to provide methods for the use of nanoparticle photosensitizer formulations based on PLGA in PDT for, but not limited to the treatment of tumors and other neoplastic diseases, dermatological disorders, ophthalmological disorders, urological disorders, arthritis and similar inflammatory diseases.
Briefly stated, present invention provides compositions, which are stable in storage, and a method of production of pharmaceutical based nanoparticulate formulations for clinical use in photodynamic therapy comprising a hydrophobic photosensitizer, poly(lactic-co-glycolic) acid and stabilizing agents. These nanoparticulate formulations provide therapeutically effective amounts of photosensitizer for parenteral administration. In particular, tetrapyrrole derivatives can be used as photosensitizers whose efficacy and safety are enhanced by such nanoparticulate formulations. It also teaches the method of preparing PLGA-based nanoparticles under sterile conditions. In one of the preferred embodiments of the present invention PLGA-based nanoparticles have a mean particle size less than 500 nm and the photosensitizer is temoporfin, 5,10,15,20-tetrakis(3-hydroxyphenyl)-chlorin (mTHPC). In another embodiment, the photosensitizer 2,3-dihydroxy-5,10,15,20-tetrakis(3-hydroxyphenyl)-chlorin (mTHPD-OH) is formulated as a nanoparticle for parenteral administration. Yet, in another embodiment preferred photosensitizer is 5,10,15,20-tetrakis(3-hydroxyphenyl)-porphyrin (mTHPP). The formulations can be used for treating hyperplasic and neoplasic conditions, inflammatory problems, and more specifically to target tumor cells.
The above, and other objects, features and advantages of the present invention will become apparent from the following description read in conjunction with the accompanying figures.
The methods of preparation of the described nanoparticle systems of the present invention provide systems that enable a drug release over several hours even in the presence of serum proteins and, therefore, are suitable for a drug transport to target cells and tissues. This is in contrast with the immediate decomposition of particles and release of photosensitizers in the prior art use of PLGA nanoparticle systems. Moreover, a high variability of drug release kinetics is obtained depending on the way the excipients are used during particle preparation.
As outlined above, questions such as sterility and freeze drying are vital to the development of nanoparticle formulations of photosensitizers. It has now been found that such PLGA-based nanoparticle photosensitizer formulations suitable for clinical applications can be prepared by an aseptic manufacturing process. Thus, present invention provides methods for the production of sterile PLGA-based photosensitizer-loaded nanoparticles of a mean particle size less than 500 nm, with the photosensitizers being chlorins or bacteriochlorins. Additionally, nanoparticle pharmaceutical formulations of the present invention are stable enough to allow freeze drying and reconstitution in an aqueous medium. Therefore present invention addresses the problem of suitable nanoparticle pharmaceutical formulations of hydrophobic photosensitizers for photodynamic therapy that meet the necessities for a parenteral administration in clinical practice.
Therapeutic uses of nanoparticle photosensitizer formulations based on PLGA in PDT include, but are not limited to dermatological disorders, ophthalmological disorders, urological disorders, arthritis and similar inflammatory diseases. More preferably, therapeutic uses of nanoparticle photosensitizer formulations based on PLGA in PDT comprise the treatment of tumor tissues, neoplasia, hyperplasia and related conditions.
The described nanoparticle systems of PLGA-based nanoparticle formulations for chlorins and bacteriochlorins for parenteral application can be prepared in the presence of reduced amounts of stabilizers (i.e. 1.0% PVA). The systems enable a drug release over several hours even in the presence of serum proteins and, therefore, are suitable for transporting a drug to target cells and tissues. The prolonged drug release enables the attachment of drug targeting ligands to the particle surface (such as antibodies) for a more advanced transport of photosensitizer to target cells and tissues.
The present invention is based in part upon the surprising discovery that during particle preparation excipients such as polyvinyl alcohol (PVA) can be used in a way, so that 1) the photosensitizer is attached by incorporation in the particle matrix, 2) is attached by adsorption to the particle matrix, 3) or is attached by incorporation in and adsorption to the particle matrix, resulting in a high variability of drug release kinetics.
In a specifically preferred embodiment of the present invention the PLGA-based nanoparticles have a mean particle size less than 500 nm and the photosensitizer is temoporfin, 5,10,15,20-tetrakis(3-hydroxyphenyl)-chlorin (mTHPC).
In another embodiment of the present invention the PLGA-based nanoparticles have a mean particle size less than 500 nm and the photosensitizer is 2,3-dihydroxy-5,10,15,20-tetrakis(3-hydroxyphenyl)-chlorin (mTHPD-OH).
In another embodiment, the PLGA-based nanoparticles have a mean particle size less than 500 nm and the photosensitizer is 5,10,15,20-tetrakis(3-hydroxyphenyl)-porphyrin (mTHPP).
The invention provides methods to prepare formulations of photosensitizer-containing nanoparticles preferably using photosensitizers of the chlorin and bacteriochlorin type. The nanoparticles prepared by the methods disclosed below have a predictable size and uniformity (in size distribution). The nanoparticles are prepared in an aseptic manufacturing process. Preferred PLGA-based nanoparticles have a mean size less than 500 nm. The term “diameter” is not intended to mean that the nanoparticles have necessarily a spherical shape. The term refers to the approximate average width of the nanoparticles.
In a preferred embodiment of the present invention the PLGA-based nanoparticles can be prepared so that the photosensitizer loading can be varied in a wide concentration range (2 to 320 μg photosensitizer per mg nanoparticles).
In a specifically preferred embodiment of the present invention the PLGA-based nanoparticles can be prepared so that the photosensitizer is attached by incorporation in the particle matrix, is attached by adsorption to the particle matrix or is attached by incorporation in and adsorption to the particle matrix, resulting in a high variability of drug release kinetics.
Drug targeting effectiveness of present nanoparticle systems may be enhanced with one or more ligands bound to PLGA-nanoparticles, maintaining the photosensitizer chemical entity by not bonding to photosensitizer molecules.
The nanoparticles of the invention may be dehydrated for improved stability on storage. The preferred method of dehydration is freeze-drying or lyophilisation. Optionally, a lyoprotectant may be used as an additive to improve the stability during the freeze-drying and during reconstitution in an aqueous medium.
In another embodiment, the present invention provides methods for the use of nanoparticle photosensitizer formulations based on PLGA in PDT, comprising the administration of the nanoparticles, their accumulation in the target tissue and the activation of the photosensitizer by light of a specific wavelength. The administration is preferably by parenteral means such as, but not limited to, intravenous injection.
Materials Used for the Preparation of the Photosensitizer-Loaded Nanoparticles Polymer
A non-limiting example of polymer to be used in the present invention is poly(D,L-lactide-co-glyeolide) PLGA, preferably characterised by a copolymer ratio of 50:50 or 75:25.
PLGA to be used for the preparations underlying the present invention was obtained from Boehringer Ingelheim (Resomer RG502H and Resomer RG504H).
Photosensitizers
The photosensitizers to be used in the present invention are preferably but not limited to tetrapyrroles of the chlorin and bacteriochlorin type. Such photosensitizers can either be derived from natural sources or by total synthesis. The total synthesis of chlorins and bacteriochlorins can be performed by first synthesizing the porphyrin and then transferring it to a chlorine or bacteriochlorin system (e.g. R. Bonnett, R. D. White, U.-J. Winfield, M. C. Berenbaum, Hydroporphyrins of the meso-tetra(hydroxyphenyl)porphyrin series as tumor photosensitizers, Biochem. J. 1989, 261, 277-280).
The chlorins and bacteriochlorins to be used with the present invention have the following preferred structure depicted in
Specifically preferred chlorins to be formulated in nanoparticles according to the present invention have the structure depicted in
The PLGA-based nanoparticles of the present invention were prepared by an emulsion-diffusion-evaporation process using an Ultra-Turrax dispersion unit. An adsorptive binding of the photosensitizer to the particle matrix, an incorporative binding into the particle matrix and a combination of adsorptive and incorporative binding to the particle matrix can be achieved. Drug loaded nanoparticles can be freeze dried in the presence of cryoprotective agents such as glucose, trehalose, sucrose, sorbitol, mannitol and the like.
The present invention is further illustrated by the following examples, but is not limited thereby.
The PLGA-based nanoparticles of the present invention were prepared by an emulsion-diffusion-evaporation process using an Ultra-Turrax dispersion unit (Ultra Turrax T25 digital, IKA, Staufen, Germany).
An amount of 500 mg PLGA (Resomer RG 502H or 504H) was dissolved in 5 mL ethyl acetate (Fluka, Steinheim, Germany). To this solution different amounts of mTHPP were added. Quantities in the range of 1 to 200 mg were under evaluation. Commonly, 50 mg of mTHPP were used.
This organic solution was added to 10 mL of a 1% polyvinyl alcohol (PVA) stabilized aqueous solution. With an Ultra-Turrax dispersion unit (17,000 rpm, 5 min) an oil-in-water nanoemulsion was formed. After this preparing step the emulsion was added to 40 mL of an aqueous PVA stabilized solution to induce the formation of nanoparticles after complete diffusion of the organic solvents into the aqueous external phase. Permanent mechanical stirring (550 rpm) was maintained for 18 h to allow the complete evaporation of ethyl acetate.
The particles were purified by 5 cycles of centrifugation (16,100G; 8 min) and redispersion in 1.0 mL water in an ultrasonic bath (5 min).
All of the aqueous solutions used for particle preparation were sterile and pre-filtered through a membrane with a pore size of 0.22 μm (Schleicher and Schull, Dassel, Germany). All of the equipment used was autoclaved at 121° C. over 20 min. All handling steps for particle preparation were performed under a laminar airflow cabinet.
Average particle size and polydispersity were measured by photon correlation spectroscopy using Zetasizer 3000 HSA (Malvern Instruments, Malvern, UK). Nanoparticle content was determined by microgravimetry.
Direct quantification procedure: The PLGA-nanoparticles were dissolved in acetone and the solution was measured photometrically at 512 nm for mTHPP to determine the content of photosensitizer. Depending upon the ratio of drug to PLGA a drug loading efficiency between 2 and 320 μg mTHPP per milligram PLGA could be achieved (
Lyophilisation of the nanoparticles can be performed according to the following protocol: For the freeze drying process trehalose was added at a concentration of 3% (m/V) to the nanoparticle samples. The samples were transferred to a freeze drier and the shelf temperature was reduced from 5° C. to −40° C. at a rate of 1° C./min. The pressure was 0.08 mbar. These parameters were held for 6 h. By increasing the temperature from −40° C. to −25° C. at 0.5° C./min the primary drying was achieved. The pressure remained unchanged. At the end of the primary drying heat ramp, a Pressure Rise Test (PRT) was performed. With termination of the primary drying the secondary drying followed by increasing the temperature at a rate of 0.2° C./min to 25° C. This temperature was held for 6 h at a pressure of 60 mT (=0.08 mbar).
Nanoparticles were prepared according to example 1a with the exception that mTHPC was used instead of mTHPP, mTHPC was photometrically quantified at 517 nm. Depending upon the ratio of drug to PLGA a drug loading efficiency between 2 and 320 μg mTHPC per milligram PLGA could be achieved.
mTHPC loaded nanoparticles were characterized as described within example 1a.
The above described standard method (example 1a) was used to prepare empty PLGA-nanoparticles. The preparation steps were performed as described for example 1a except for the addition of the photosensitizer mTHPP.
In the next step a PVA-stabilized mTHPP solution was prepared. Therefore, 25 mg mTHPP was solved in 5 mL ethyl acetate and afterwards 10 mL of a 1% aqueous PVA solution was added. With an Ultra-Turrax dispersion unit an emulsion was prepared. The emulsion was added to 40 mL PVA solution (1%). Permanent mechanical stirring (550 rpm) was maintained for 18 h to allow the complete evaporation of ethyl acetate.
A volume of the PLGA-nanoparticle suspension corresponding to 10 mg nanoparticles was centrifuged (16,100G; 8 min) and the supernatant was discarded. The nanoparticles were redispersed in the PVA-stabilized mTHPP solution using an ultrasonic bath (5 min).
The mixture was agitated (Thermomixer comfort, Eppendorf, Hamburg, Germany) for 18 h (500 rpm, 20° C.) to achieve adsorption equilibrium of mTHPP to the particle surface. The nanoparticles were purified as previously described.
Depending upon the ratio of drug to PLGA a drug loading efficiency between 2 and 80 μg mTHPP (according to the standard protocol typically 20 μg) per milligram PLGA could be achieved.
mTHPP loaded (adsorbed) nanoparticles were characterized and lyophilized as described within example 1a.
Nanoparticles were prepared according to example 1e with the exception that mTHPC was used instead of mTHPP. mTHPC was photometrically quantified at 517 nm.
Depending upon the ratio of drug to PLGA a drug loading efficiency between 2 and 80 μg mTHPC (according to the standard protocol typically 20 μg) per milligram PLGA could be achieved.
mTHPC loaded (adsorbed) nanoparticles were characterized and lyophilized as described within example 1a.
PLGA nanoparticles were prepared according to example 1a. The resulting nanoparticles were washed with aqueous 5% (m/V) PVA solution instead of purified water in order to displace the adsorptive bound mTHPP from the nanoparticle surface. After 3 cycles of washing with PVA solution the nanoparticles were further purified by repeated centrifugation and redispersion in purified water.
Depending upon the ratio of drug to PLGA a drug loading efficiency between 15 and 80 μg mTHPP (according to the standard protocol typically 50 μg) per milligram PLGA could be achieved.
mTHPP loaded (incorporated) nanoparticles were characterized and lyophilized as described within example 1a.
Nanoparticles were prepared according to example 1e with the exception that mT PC was used instead of mTHPP. mTHPC was photometrically quantified at 517 nm.
Depending upon the ratio of drug to PLGA a drug loading efficiency between 15 and 80 μg mTHPC (according to the standard protocol typically 50 μg) per milligram PLGA could be achieved.
mTHPC loaded (incorporated) nanoparticles were characterized and lyophilized as described within example 1a.
To show the cellular uptake and cell adhesion, respectively, and the intracellular distribution of the PLGA-based nanoparticles, the confocal laser scanning microscopy was used. HT29 cells were cultured on glass slides (BD Biosciences GmbH, Heidelberg) and incubated with the nanoparticulate formulation for 4 h at 37° C. Following, the cells were washed twice with PBS and the membranes were stained with Concanavalin A AlexaFluor350 (50 μg/ml; Invitrogen, Karlsruhe) for 2 min. Cells were fixed with 0.4% paraformaldehyde for 6 min. After fixation, the cells were washed two times and embedded in Vectashield HardSet Mounting Medium (Axxora, Grünberg). The microscopy analysis was performed with an Axiovert 200 M microscope with a 510 NLO Meta device (Zeiss, Jena), a chameleon femtosecond or an argon ion laser and the LSM Image Examiner software. The green fluorescence of the PLGA based nanoparticles leading from incorporated Lumogen Yellow® (BASF; Ludwigshafen) and the red autolluorescence of the photosensitizer 5,10,10,20-tetrakis(3-hydroxyphenyl)-porphyrin (mTHPP) was used to determine the distribution.
(
Cell Uptake and Cell Adhesion, Respectively, of PLGA-Based Nanoparticles with the Photosensitizer 5,10,15,20-tetrakis(3-hydroxyphenyl)-chlorin (mTHPC)
(
The samples listed in Table 1 were tested for intracellular uptake and phototoxicity of mTHPC-PLGA-nanoparticles.
All cell samples were incubated with a dye concentration of 3 μM mTHPC in the medium (RPMI1640) for 1 h, 3 h, 5 h, 24 h in Jurkat cell suspensions
Phototoxicity of different mTHPC loaded PLGA nanoparticles on Jurkat cells after different incubation times was assessed with the Trypan blue test and apoptotic change of the cell shape. Experiments were performed with a 660 nm LED light source, an exposure time of 120 s and a light dose of 290 mJ/cm2.
Experiments to quantify the intracellular uptake of different mTHPC loaded PLGA nanoparticles were also performed.
After incubation the cells were counted using a haemocytometer, washed (PBS, 400 g, 3 min, 2×) and the cell pellet was stored frozen overnight at −20° C. to disrupt the cell membranes.
From these cells the mTHPC was extracted in ethanol using ultrasound.
The mTHPC concentration in the ethanol extract was determined via fluorescence using a standard fluorescence series. For the calculation of intracellular concentration the diameter of the cells was assumed to be 10 μm (3 measurements).
All three PLGA-nanoparticles transport mTHPC into the cells. The transport into the cells occurs in a faster way, when the mTHPC is incorporated in the NPs.
After 5 h incubation all NPs cause a high phototoxicity.
Having described preferred embodiments of the invention with reference to the accompanying drawings, it is to be understood that the invention is not limited to the precise embodiments, and that those skilled in the art can effect changes and modifications without departing from the scope of the invention as defined in the appended claims.
This application claims the benefit and priority of U.S. Provisional Application Ser. No. 61/285,895 filed Dec. 11, 2009, entitled “NANOPARTICLE CARRIER SYSTEMS BASED ON POLY(DL-LACTIC-CO-GLYCOLIC ACID) (PLGA) FOR PHOTODYNAMIC THERAPY (PDT)” by Klaus Langer et al., which is incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 61285895 | Dec 2009 | US |
