WO2007120504A2

WO2007120504A2 - Targeted charge-reversal nanoparticles for nuclear drug delivery

Info

Publication number: WO2007120504A2
Application number: PCT/US2007/008146
Authority: WO
Inventors: Youqing Shen; Peisheng Xu
Original assignee: University Of Wyoming
Priority date: 2006-03-31
Filing date: 2007-04-02
Publication date: 2007-10-25
Also published as: WO2007120504A3

Abstract

Novel compounds are described that have a negative charge at high pH and are readily hydrolysable at low pH to have a positive charge. The negatively charged molecules have a low interaction rate in the body whereas the positively charged molecules have a high interaction profile. The compounds are useful as drug and gene delivery carriers in the body.

Description

TARGETED CHARGE-REVERSALNANOPARTICLESFORNUCLEARDRUG

DELIVERY

Background of the Invention

The invention relates generally to compositions having charges that vary with pH and, more specifically, to compounds containing both amine and carboxylic acid moieties that are negatively charged at high pH and positively charged at low pH.

Most of cancer chemotherapy drugs such as anthracyclines and cisp latin target nuclear DNA to cause DNA damages and/or topoisomerase inhibition to induce the cell death (apoptosis)/¹'²⁵ Drag resistant cancer cells, however, have overexpressed membrane- associated multidrug resistance mechanisms to transport drugs out of the cells^p>4] and many intracellular drug-resistance mechanisms to limit the access of drugs to the nucleus.¹-⁵'⁶¹ Consequently, only a small percentage of drugs delivered into the cytosol finally reach the nucleus in drug-resistant cells. For example, less than 1% of the cisplatin molecules that enter the cell actually bind the nuclear DNA P^ Thus, a drug carrier capable of localizing and directly releasing drugs into the nucleus would circumvent the membrane-associated multidrug resistance and intracellular drug resistance mechanisms to deliver a high concentration of drug to the nucleus.

Polymer micelles and nanoparticles^[7"n] have been found to carry drugs preferentially to cancerous tissues via the enhanced permeation and retention (EPR) effect/¹²' ¹^ and bypass the PGP-based multidrug resistance in the cell membrane/¹⁴¹ but the nanoparticles developed to date were found retained in several cytoplasmic organelles including lysosomes rather than the nucleus.^[I5^ There are many reports using nuclear localization peptides (NLPs) — short highly positively charged peptides mat actively transport large proteins across the nuclear membrane — to localize drug molecules from the cytosol to the nucleus.¹¹⁶' ^17] A cationic polymer, polyethyleneimine (PEI), has been used extensively in nonvirual gene delivery. It can carry DNA across the cell membrane, then harness the molecular motors to actively move along the microtubule network to the peripheral of the nucleus, and finally enter the nucleus/^{1 s'20]} which may or may not depend on the breakdown of the nuclear envelope in cell-dividing. ^[20"22] NLPs and PEI, however, are highly positive charged at the physiological pH. Positively charged macromolecules or colloidal particles can cause severe serum inhibition and are rapidly cleared from the plasma compartment. ^[23"25] Thus, drug carriers using PEI or NLS would have a rapid plasma clearance, making them unlikely to reach their targeted cancer tissues other than the liver.

Particles, polymers and peptides carrying primary or secondary amines are positively charged and have many applications. Particularly, these polymers, peptides or particles can interact with negatively charged cell membranes, and thus can be rapidly internalized by cells. Accordingly, they can be very efficient drug delivery carriers. Furthermore, amine- based polymers such as polyethyleneimine (PEI) efficiently disrupt lysosomes and can guide DNA molecules and fragments to nuclei, thus can be used for gene delivery. However, positively charged polymers, particles or peptides are very toxic and they can immediately interact with cells or other components in the blood compartment. They cannot, therefore, be injected intravenously. A preferred regime would be to activate the cationic charges only in cancerous tissues or their intracellular compartments. Amides with neighboring carboxylic acid groups have a pH-dependent hydrolysisJ³¹^ Nanoparticles with a negative-to-positive charge^'reversal PEI-outer layer triggered by the solid tumor extracellular- (pH <7, ^{fl3> 26]}) or lysosomal (pH=Φ-5,^[27]) acidity will have applicability for nuclear drag delivery. Negatively charged polymers have little interaction with the blood components and have been used extensively in vzvo.^[28'30]

Summary of the Invention

The present invention discloses a technique that can preserve the amine groups as negatively charged groups that have no or low interactions with cells and low toxicity. These groups are stable at neutral pH but rapidly decompose and convert back to amine groups at pH less than 7. This technique has many applications including their development as drug or gene delivery carriers.

In a preferred embodiment of the invention, nanoparticles with a negative-to-positive charge^'reversal outer layer, such as PEI, that is triggered by the solid tumor extracellular or lysosomal acidity for nuclear drug delivery.

Brief Description of the Drawings

Fig. 1 is a chart of the pH-dependent hydrolysis of the amide bond shown in Scheme 1. Fig. 2 is a chart of the zeta-potential as a function of time at pH 5 of micelles made from PCL-PEI-An.

Fig. 3 is a chart of the zeta-potential as a function of time at pH 6 of micelles made from PCL-PEI-An.

Fig. 4 is a chart of the zeta-potential as a function of time at pH 7.4 of micelles made from PCL-PEI-An.

Fig. 5 is a graph of the overall hydrolytic kinetics of the amides in the PLL/amide at different acidities at 37 ⁰C.

Fig. 6 is a graph of the ξ-potential of PLL/amide as a function time at different pHs.

Fig. 7 is a graph of the overall hydrolytic kinetics of the amides in the PCL- PEI/amide-FA at different acidities at 37⁰C.

Fig. 8 is a graph of the ξ-potential of the PCL-PEl/amide nanoparticles at 37 ⁰C as a function of time at different acidities.

Fig. 9 is a graph of the SKO V-3 cellular uptake of TCRNs/DOX at different pHs. DOX dose: lμg/mL; 1 h incubation at 37 C; results are presented as a mean of three experiments, * P<0.05.

Fig. 10 is a graph of SKOV-3 cellular uptake of DOX-loaded TCRNs and charge reversible nanoparticles (CRNs) made from PCL-PEl/amide shown in Scheme 2 but without the folic acid moieties. DOX dose: 0.5 μg/mL; pH 7.4; 2 h incubation; results are presented as a mean of three experiments and standard deviation, * P< 0.05.

Fig. 11 is a graph of the hemolytic activity of TCRNs on RBCs at pH of 6 and 7.4 as a function of TCRN concentration. (1 h incubation at 37 ⁰C).

Fig. 12 is a graph of the cytotoxicity of DOX and TCRNs/DOX to SKOV-3 ovarian cancer cells as a function of DOX dose; results are presented as a mean of four experiments and standard deviation.

Description of the Invention

The amides with neighboring carboxylic acid groups are pH sensitive. They are stable at neutral pH but decompose at acidic pHs, as shown in Scheme 1. The generated amines carry cationic charges.

Scheme 1. Hydrolysis of a model amide with a neighboring carboxylic group

Negative to positive charge reversible polymers

Primary and/or secondary amine-containing polymers or peptides or other amine- containing biopolymers can be made into negatively charged by the reaction with the anhydrides as scheme 1. Once at acidic pHs, the amides hydrolyze and become amine groups, as shown in Figure 1. These polymers or peptides turn into positively charged.

Negative to positive charge reversible nanoparticles

Nanoparticles or microparticles with outer layers containing amide-anhydride groups shown in Scheme 1 carry anionic charges at neutral pH but become positively charged at acidic pHs. Scheme 2 shows one example. Anionically modified poly(caprolactone)-block- polyethyleneimine with about 1/3 of its amines reacted with an anhydride (PCL-PEI-An) is synthesized. The block copolymer forms micelles with about 100 nm in diameter. At pH 7.4, the micelles are always negatively charged (Fig. 4), but pH 6 and 5, the micelles immediately positively charged (Figs. 2 and 3, respectively).

Scheme 2. The synthesis of anionically modified PCL-PEI-An Fabrication of nanoparticϊes-loaded with DQX (TCRNs/DOX):

Doxorubicin (DOX) hydrochloride salt (0.5 mg) was dissolved in 2 ml of DMSO and stirred for 10 min. Triethylamine (10 μL) was added and stirred for another 1 h. PCL- PEI/amide-FA (2.5 mg) was dissolved in 5 ml of DMSO. The two DMSO solutions were mixed together and stirred for 1 h. The mixture was loaded into a dialysis bag (Spectra Por - 7, MWCO 3,500) and dialyzed against with 2 L PBS. The free DOX was removed by filtering the solution through a 0.45 μm filter. The DOX drug loading was analyzed by measuring the UV absorbance at 486 nm in DMSO/CHC1₃. The encapsulation efficiency was 88%, and the loading content was 14.6%. TCRNs without DOX were fabricated similarly except without adding DOX. The typical anhydrides or acids that can be used are shown as:

R1 , R2, R3, R4 can be any groups or H

EXAMPLE l

An example of charge-reversal poly(L-lysine) (PLL) amide is shown in Scheme 2. It can be made by the reaction of PLL with the corresponding anhydride. The PLL/amide was dissolved in buffers at pH 7.4, 6 or 5 and the hydrolysis of the amide was monitored by NMR. At pH 7.4, about 20% the amide hydrolyzed even after 80 h, but at pH 5, all the amide bonds hydrolyzed within 1O h. The hydrolysis at pH 6 was slower than that at pH 5 but much faster that at pH 7.4 (Figure 5). The corresponding charge reversal of the PLL/amide was determined by measuring its ξ-potentials using Nano-ZS (Malvern). PLL itself had a ^-potential of 15±2 mV independent of pH. The PLL/amide gradually became positively charged with a ξ- potential of about +10 mv at pH 5 and + 5 mv at pH 6 (Figure 6). The PLL/amide remained negatively charged at pH 7.4 even after 72 h, which makes it suitable for in vivo applications.

Scheme 2: Charge-reversal polylysine

EXAMPLE 2

Liposomes, nanoparticles or micelles or microparticles with outer layers containing hydrolysable amides are neutral or negatively charged at neutral pH but become positively charged at acidic pHs. Scheme 3 shows one example. A model polymer, polycaprolactone (Mn =3800)-block-PEI (Mn =1800) (PCL-PEI) was synthesized. Its PEI block reacted with 1,2-cyclohexanedicarboxylic anhydride to convert all or part of the primary and secondary amines into their amides (PCL-PEI/amide) (Scheme 3). It forms micelles of about one hundred nanometers.

Scheme 3. The structure poly(ε-caprolactone)-block-PEI with the amines converted to their amides ( PCL-b-PEI/amide-FA)

The degree of the amidization was optimized and it was found that the PEI block with 20% of the primary and secondary amines converted to the amides was optimal in terms of the charge reversal kinetics of the resulting nanoparticles (Scheme 2). If all the primary and secondary amines were reacted to their amides, the resulting nanoparticles could not rapidly become highly negative charged at low pH. The folic acid moieties were also conjugated to the PEI block to form PCL-b-PEI/amide-FA (Scheme 2) for folate receptor targeting. ^[32] It was estimated from the NMR spectra that there was 0.79 folic acid molecule per PCL-PEI chain on average. The PCL-PEI/amide-FA formed nanoparticles of about 210 nm in diameter in water. The nanoparticles were about 120 nm in diameter if loaded with 14.6 wt% DOX. Transmission electron microscopy (TEM) images showed that these nanoparticles were spherical.

The hydrolysis kinetics of the amides in the PCL-PEI/amide was determined by dispersing the nanoparticles in solution at pH 7.4, 6.0 or 5.0. The concentration of free 1,2- czs-cyclohexanedicarboxylic acid in the solution hydrolyzed from the PEI/amide was determined by NMR using DMF as the internal reference, and the percent of the unhydrolyzed amides was calculated accordingly. Figure 7 shows the overall hydrolysis kinetics of the amides of the primary and secondary amines in the PEI block. The amides hydrolyzed about 70% at pH 5.0 and 40% at pH 6.0 in 2 h. At pH 7.4, only about 25% amide bond hydrolyzed even after 24 h. The amides hydrolyzed more than 50% and 75% at pH of 6.0 and 5.0 respectively, after 24 h. Accordingly, the charge reversal of the PCL-PEI/amide micelles was determined by measuring their ξ-potentials at different acidities (Figure 8). The micelles of PCL-PEI/amide had a ξ-potential of about -20 mV at pH 7.4 even after more than 60 h, indicating that they were always negatively charged due to the presence of -COOH groups. At pH 5, they immediately became highly positively charged, and gradually reached a ξ-potential of about +50 mV in about 10 h. At pH 6, the ξ-potential was about +8 mV. For comparison, the micelles of PCL-PEI were always positively charged. Their ξ-potential was +36.1 mV at pH 5, +18.4 mV at pH 6 and +17.5 mV at pH 7.4. Thus, the PCL-PEI/amide micelles were indeed charge reversal: they were negatively charged at the physiological pH and thus suitable for in vivo applications. Once localized in solid tumors or lysosomes, the PEI/amides are expected to hydrolyze and recover the PEI, and the micelles become positively charged. With the folic acid targeting groups, the micelles are named targeted charged reversal nanoparticles (TCRNs) (Scheme 2).

EXAMPLE 3

The nuclear localization of charge-reversal nanoparticles was demonstrated using the PCL-PEI/amide nanoparticles by observing the SKOV-3 cells cultured with particles loaded with DOX or PKH26 fluorescent dye using confocal microscopy. After 12 h incubation with SKOV-3 cells, the nanoparticles loaded with PKH26 localized in some nuclei but mostly associated with nuclear membranes. After 24 h incubation with the SKOV-3 cells, many nanoparticles/PKH26 appeared in the nuclei. This proved that, in contrast to the conventional nanoparticles with PEG corona, which are retained in the lysosomes and other subcellular compartments, the charge reversal nanoparticles could indeed enter the nuclei of the cancer cells, which can potentially enhance the efficiency of the drug.

EXAMPLE 4

The cellular internalization of TCRNs loaded with DOX (TCRNs/DOX) was measured using flow cytometry (Figure 9). The percentage of DOX-positive cells cultured with TCRNs/DOX was significantly higher than that cultured with free DOX under the same conditions at pH7.4. This is a significant improvement compared with reported results in which the cellular uptake of DOX in drug carriers was generally slower than that of free DOX; free DOX enters cell via a rapid diffusion process, while drug carriers enter cells via the slower endocytosis process.¹³³¹ Figure 3 also shows that TCRNs/DOX entered cells faster at pH 6 than at pH 7.4. This is agreeable with the result in Figure 2 that some positive charges were generated on the TCRNs at pH 6. Positive charges promote the cellular internalization via electrostatically-adsorptive endocytosis/³⁴' ^35] Thus, it is expected that at the acidic solid tumor interstitium, TCRNs would regenerate some positive chares (Figure 2) and promote their cellular uptake.

EXAMPLE 5

The effectiveness of the targeting group folic acid on the TCRNs in binding folate receptors and promoting the cellular uptake was also evaluated using SKO V-3 ovarian cancer cells, which are known to overexpress folate receptors,¹³²¹ as shown in Figure 10. TCRNs were internalized much faster into SKOV-3 ovarian cancer cells than the charge reversal nanoparticles made from the same PCL-PEI/amide but without the folic acid moieties (CRNs). This indicates that TCRNs indeed effectively target the folate receptor- overexpressing cancer cells.

EXAMPLE 6

After internalized, the TCRNs must localize in lysosomes to regenerate the PEI layer. The intracellular trafficking of the nanoparticles was analyzed using confocal scanning laser fluorescent microscopy. Most internalized TCRNs were observed to be localized in lysosomes. Some TCRNs were not associated with lysosomes, suggesting that these TCRNs might already escaped from lysosomes within 2 h incubation. The ability of the TCRNs to escape from lysosomes was evaluated by measuring their hemolysis activity. Hemolysis of red blood cells (RBCs) has been used as a measure of a drug carrier's ability to rupture lysosomes.¹³⁶¹ The hemolysis of TCRNs was evaluated at pH 6 rather than at the lysosomal pH (4-5) because this low pH caused a significant fraction of RBCs lysed. Figure 11 shows that at pH 6 TCRNs lysed RBCs even at very low concentrations. This is in agreement with the results in Figure 8. The hydrolysis of PEI/amide at pH 6 produced amine groups carrying positive charges, causing TCRNs to adsorb on the RBCs and rupture them. One thus can expect that TCRNs would more efficiently rupture lysosomes, where the pH is 4-5 and TCRNs quickly become fully positively charged. This likely explains why some of TCRNs were not associated with lysosomes.

Figure 11 also shows that at the neutral pH, basically no RBC hemolysis occurred at TCRN concentrations less man 10 μg/mL, indicating that TCRNs had little interaction with RBCs. Thus, the nanoparticles are suitable for in vivo applications/³⁷¹

EXAMPLE 7

The nuclear localization of TCRNs was monitored by observing the SKO V-3 cells cultured with TCRNs loaded with DOX or PKH26 dye using confocal microscopy. After 8 h incubation with SKO V-3 cells, TCRNs/DOX were found very close to or even associated with the nuclear membrane. To further probe the nuclear localization of TCRNs at longer times, the nanoparticles were loaded with PKH26 (TCRNs/PKH26) instead of DOX because DOX released from the TCRNs could enter the nucleus and might produce misleading results. In addition, the cells having DOX in their nuclei died very quickly. PKH26 is a cell membrane dye, which preferentially binds the cell membrane. Thus, it can only be delivered to the nucleus by the TCRNs. It shows no apparent toxic effect to cells. At 12 h, the TCRNs/PKH26 were observed to be localized in some nuclei but mostly associated with nuclear membranes. After 24 h incubation with the SKOV-3 cells, many TCRNs/PKH26 appeared in the nuclei. This proved that, in contrast to the conventional nanoparticles with PEG corona, which are retained in the lysosomes and other subcellular compartments^[15], TCRNs could indeed enter the nuclei of the cancer cells.

EXAMPLE 8

The in vitro cytotoxicity of DOX encapsulated in TCRNs (TCRNs/DOX) was evaluated by measuring the IC₅₀ using the MTT assay (Figure 12). The IC₅₀ of free DOX was about 1.5 μg/mL, while it decreased to 0.23 μg/mL when it was encapsulated in TCRNs (Figure 8). This is different from most other reported nanoparticles as DOX carriers, in which DOX in the nanoparticles showed a lower cytotoxicity than free DOX.¹-^38"40-¹ This comparison suggests that the TCRNs could efficiently cross the cell membrane, escape from the lysosomes, localize and deliver DOX in the nucleus, resulting in a greater cytotoxicity. In summary, we demonstrated that TCRNs made from folic-acid-functionalized PCL- b-PEI/amide are negatively charged in the neutral solution but quickly become positively charged at pH 6, and highly positively charged at pH 5. The hydrolysis kinetics indicate that amides with β-carboxylic acid can hydrolyze in acidic conditions to regenerate the amines, giving rise to an anionic to cationic charge reversal. These recovered amines carry cationic charges, which can effectively enhance the cellular uptake of the nanoparticles, and thereafter direct the TCRNs localize in the nucleus. In vitro experiment shows that TCRNs/DOX are more effective in killing SKO V-3 cancer cells than free doxorubicin.

The foregoing description and drawings comprise illustrative embodiments of the present inventions. The foregoing embodiments and the methods described herein may vary based on the ability, experience, and preference of those skilled in the art. Merely listing the steps of the method in a certain order does not constitute any limitation on the order of the steps of the method. The foregoing description and drawings merely explain and illustrate the invention, and the invention is not limited thereto, except insofar as the claims are so limited. Those skilled in the art who have the disclosure before them will be able to make modifications and variations therein without departing from the scope of the invention.

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Claims

We claim:

1. A method for protecting primary, secondary or argino- amines by attaching hydrolysable amides.

2. The method as defined in claim 1, wherein the amides are stable at the neutral or basic pHs but hydrolyze at pH lower than 7.

3. The method as defined in claim 1 , wherein the amines comprise polymers, pepteides, proteins or any particle surface.

4. A composition, comprising peptides, proteins or other biopolymers that are negatively charged or neutral at pH higher than 7, but become positively charged at pH lower than 7.

5. A composition as defined in claim 4, further comprising a bioactive agent and wherein the peptide, protein or other biopolymer hydrolyses at a delivery site of the bioactive agent to release the bioactive agent at the delivery site.

6. A composition, comprising particles that are negatively charged or neutral at pH higher than 7, but become positively charged at pH lower than 7.

7. A composition as defined in claim 6, further comprising a bioactive agent and wherein the bioactive agent is contained inside particles and wherein at least a portion of the particles hydrolyse at a delivery site of the bioactive agent to release the bioactive agent at the delivery site.