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Charge-Reversal Drug Conjugate for Targeted Cancer Cell Nuclear Drug Delivery By Zhuxian Zhou, Youqing Shen,* Jianbin Tang, Maohong Fan, Edward A Van Kirk, William J Murdoch, and Maciej Radosz

leaky blood capillaries and preferentially deliver drugs to cancer tissues by the DNA-toxin anticancer drugs target nuclear DNA or its associated enzymes to enhanced permeability and retention elicit their pharmaceutical effects, but cancer cells have not only membraneeffect.[6] Furthermore, carriers with such associated but also many intracellular drug-resistance mechanisms that limit targeting groups as folic acid (FA) can their nuclear localization. Thus, delivering such drugs directly to the nucleus specifically target and be efficiently interwould bypass the drug-resistance barriers. The cationic polymer poly(L-lysine) nalized by tumor cells that overexpress their receptors by receptor-mediated endocytosis, (PLL) is capable of nuclear localization and may be used as a drug carrier for and deliver drugs into the cytosol.[7] The nuclear drug delivery, but its cationic charges make it toxic and cause cytoplasmic drug delivery by polymer–drug problems in in-vivo applications. Herein, PLL is used to demonstrate a pHconjugates,[8] and micelles or nanoparticles[9] triggered charge-reversal carrier to solve this problem. PLL’s primary amines can circumvent membrane-associated drug are amidized as acid-labile b-carboxylic amides (PLL/amide). The negatively resistance. Therefore, by increasing the bioavailability of drugs at sites of action charged PLL/amide has a very low toxicity and low interaction with cells and, and bypassing the multidrug resistance, therefore, may be used in vivo. But once in cancer cells’ acidic lysosomes, the drugs in these carriers have shown theraacid-labile amides hydrolyze into primary amines. The regenerated PLL peutic advantages that include enhanced escapes from the lysosomes and traverses into the nucleus. A cancer-cell efficacy against resistant tumors and fewer targeted nuclear-localization polymer–drug conjugate has, thereby, been side effects.[10] developed by introducing folic-acid targeting groups and an anticancer drug Many anticancer drugs, such as anthracylines, camptothecin (CPT), and cisplatin, camptothecin (CPT) to PLL/amide (FA-PLL/amide-CPT). The conjugate are DNA-toxins, which target nuclear DNA efficiently enters folate-receptor overexpressing cancer cells and traverses to to cause DNA damage or inhibit topoitheir nuclei. The CPT conjugated to the carrier by intracellular cleavable somerase involved in DNA replication to disulfide bonds shows much improved cytotoxicity. induce cell death (apoptosis).[11] They thus have to localize in the nucleus to elicit their pharmacological responses. However, drug-resistant cells have many intracellular drug-resistance 1. Introduction mechanisms to limit the access of cytosolic drugs to the nucleus by such mechanisms as drug metabolism and detoxification, drug Nanometer-sized drug carriers,[1] which include polymer–drug sequestering to acidic compartments,[12] drug deactivation, and [2] [3] [4] conjugates, dendrimers, liposomes, polymer micelles, and binding to cytosolic macromolecules.[13] Drug-resistant cells also nanoparticles[5] have been extensively explored in drug delivery for have P-glycoprotein overexpressed on the membranes of cancer chemotherapy. They can easily extravasate from the tumor’s cytoplasmic organelles and the nuclear envelope membrane, which actively regulates the intracellular drug sequestration and [*] Prof. Y. Shen, Dr. J. Tang outwards transport of drugs from their subcellular targets such as Center for Bionanoengineering and State Key Lab of Chemical the cell nucleus.[14] As a result, only a small percentage of drugs Engineering delivered into the cytosol finally reaches the nucleus in drugDepartment of Chemical and Biochemical Engineering resistant cells. For example, less than 1% of the cisplatin molecules Zhejiang University, Hangzhou, 310027 (P. R. China) that enter the cell actually bind to nuclear DNA.[15] Therefore, the E-mail: [email protected]; [email protected] cytoplasmic drug delivery for DNA-toxins is not sufficient. Prof. Y. Shen, Z. Zhou, Prof. M. Fan, Prof. M. Radosz Department of Chemical and Petroleum Engineering Thus, we postulate that a DNA-toxin carrier that can directly University of Wyoming, Laramie, WY 8207 (USA) localize and release the drug in the vicinity of its target—the E. A Van Kirk, Prof. W. J Murdoch nuclear DNA—would circumvent the membrane-associated Department of Animal Science multidrug resistance and intracellular drug-resistance mechanUniversity of Wyoming, Laramie, WY 8207 (USA) isms, and efficiently induce cell apoptosis. Drug-delivery vehicles developed to date, however, mainly retain and release drugs in the DOI: 10.1002/adfm.200900825 3580

ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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cytoplasmic organelles rather than the nucleus.[16] They are internalized by cells through endocytosis into clathrin-coated vesicles that fuse to form early endosomes (pH 6–7.4), and then become late endosomes (pH 5.5–6.0), and eventually lysosomes (pH 5.0), where they release the drug.[17] Even though there are many in-vitro studies using nuclear-localization signals (NLSs) to deliver various small or large molecules from the cytosol to the nucleus,[18] NLSs cannot be used in vivo for targeted delivery because they are non-specific in the body and can distribute in all tissues, even across the blood–brain barrier.[19] Cationic polymers such as poly(ethyleneimine) and poly(L-lysine) (PLL) have been extensively explored for gene delivery.[20] They can promote the entry of materials into the nucleus.[21] Thus, drugs conjugated to cationic polymers might be delivered to the nucleus. However, cationic polymers or colloidal particles in the bloodstream have strong non-specific cellular uptake, can cause severe serum inhibition, and can be rapidly cleared from the plasma compartment, and thus cannot be used in vivo as drug carriers.[22] An ideal scenario would be that the cationic polymers’ positive charges are masked during in the blood circulation, but regenerated once inside the cancer cells for nuclear localization. Amides with b-carboxylic acid groups are acid labile.[23,24] We used this type of amide to reversibly modify the amine groups and developed charge-reversal nanoparticles for nuclear drug delivery. We found that the nanoparticles could localize in the nucleus, and the drug encapsulated in the nanoparticle had elevated cytotoxicity to cancer cells.[25] The nanoparticles, of about 100 nm in diameter,

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Scheme 1. The targeted charge-reversal PLL conjugate structure and its acid-triggered charge reversal (a) and nuclear drug delivery (b). The conjugate accumulates in cancer tissues by the enhanced permeation and retention effect (A); its folic acid targeting group binds the receptor (B1); it is taken up by receptor-mediated endocytosis (C), transferred into an endosome (D), and then a lysosome (E); the labile amides hydrolyze at the lysosomal pH (4–5) to regenerate the PLL as the carrier. The regenerated PLL carrier ruptures the lysosomal membrane to escape into the cytosol (F) and traverse into the nucleus (G) and releases the carried drug there (H). In acidic solid tumor tissues, the PLL may also regenerate and thus is adsorbed on the cell membrane, triggering electrostatically adsorptive endocytosis (B2)

however, were too big to go through the nuclear pores, which only transport particles of a maximum 39 nm.[26] Therefore, the nanoparticles had to wait until the nuclear membrane collapsed during the mitosis to localize in the nucleus. Active nuclear localizing drug carriers with a size smaller than 39 nm may easily go through the nuclear pores and be more efficient for nuclear drug delivery. Polymer–drug conjugates are generally less than 10 nm. Therefore, the aim of this work is to demonstrate a cancertargeted nuclear-localizing polymer–drug conjugate for cancer nuclear drug delivery using the cationic polymer PLL as an example (Scheme 1). The PLL’s primary amine groups were amidized as acid-labile b-carboxylic acid amides (PLL/amide), thus the PLL/amide was negatively charged at neutral pH, which inhibited the PLL’s non-specific cellular uptake and clearance from the blood compartment. Its tumor targeting and cellular uptake were enhanced by a targeting group FA. Once in cancer cells’ lysosomes, the amides quickly hydrolyzed to regenerate the positively charged PLL, which then escaped from the lysosomes and traversed to the nucleus. Camptothecin (CPT) conjugated to the PLL/amide by a disulfide bond shows much enhanced cytotoxicity over free CPT.

2. Results and Discussion 2.1. Design and Synthesis of the Nuclear-Localizing Conjugate The conjugate for in-vivo tumor-targeted nuclear drug delivery should have low interactions with the blood serum and red blood cells (RBCs) to ensure a long blood circulation time, be able to bind tumor cells and be quickly taken up once in the tumor, become positively charged in an acidic lysosome, and then quickly escape from the lysosome, traverse to the nucleus, and finally release the drug (Scheme 1b). Accordingly, a charge-reversal conjugate was designed using a synthetic peptide PLL as the drug carrier because of its biodegradability[27] (Scheme 1a). The primary amines in its residues were amidized, to form acid-labile amides, to inhibit its interactions with cells before it reaches tumor tissues, but once at the lysosomal pH, the amides quickly hydrolyzed to regenerate the PLL and regain its lysosomal-escaping and nuclear-localization abilities. FA groups were conjugated to the PLL/amide for tumor targeting and cellular uptake because folic receptors are overexpressed in many malignant tumor cells but in few normal cells.[7] The cell nucleus is neutral, and thus lysosomal acid-sensitive drug linkers cannot be used for nuclear drug release. Thus, the drug CPT was anchored to the carrier by a glutathione (GSH)-reactive disulfide bond, which is cleavable in the strongly reducing intracellular environment ((0.5–10) 103 M GSH) but stable in the blood ((20–40) 106 M GSH).[28]


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but should quickly hydrolyze to regenerate the cationic PLL at the tumor extracellular pH (pH < 7) or the endosomal/lysosomal pH (pH 4–5) for cellular uptake, endosomal/ lysosomal escape, and nuclear localization (Scheme 1b). The b-carboxylic acid-amide structures were first optimized by comparing the acid-triggered hydrolysis of the amides resulting from the different anhydrides (Scheme 3) at a pH of 7.4, 6.0, and 5.0 monitored by 1H NMR spectroscopy. The chemical shift of the methylene proton in CH2NH2 changed from 2.9 to 3.1 ppm after the amine was amidized. The typical intensity change of the two peaks during the hydrolysis of PLL/DM at pH 6.0 is shown in Figure 3. With hydrolysis the intensity of the CH2-amide peak decreased while that of the CH2-amine increased. The intensity ratio of the two peaks was used to calculate the percentage of amide hydrolyzed. The hydrolytic rates of the PLL/ Scheme 2. Synthesis of the targeted charge-reversal conjugate FA-PLL/DCA-CPT. amide made from the three anhydrides at different pH are shown in Figure 4 and summarized in Table 1. All the amides were acid-labile catalyzed The synthesis of the FA-PLL/amide-CPT is shown in Scheme 2. by a neighboring carboxylic acid.[23] The amides hydrolyzed PLL with about 50 repeat units (determined by 1H NMR analysis) [29] was synthesized according to the literature. quickly at an acidic pH (e.g., pH 5) but slowly as the pH increased. FA was first The amides were stable in basic conditions (pH 8.5). At the introduced to PLL by the reaction of its N-(3-dimethylaminoprosame pH, the hydrolysis rates of the amides made from the three pyl)-N0 -ethylcarbodiimide hydrochloride (EDC)-activated g-caranhydrides were in the order of PLL/DM > PLL/DCA > PLL/TM. boxylic acid with the PLL primary amine. Both UV and NMR were At pH 6.0, the time needed for 50% of the amide to hydrolyze was used to determine the average number of FA molecules attached to about 10 min for PLL/DM, 6.5 h for PLL/DCA, and 30.5 h for PLL/ the PLL.[30] Thiolated CPT (CPT-SH) was then attached to the PLL TM. by a heterobifunctional coupling reagent N-succinimidyl 3-(2Each b-carboxylic amide in the PLL/amide has a b-carboxylic pyridyldithio) propionate (SPDP). Subsequently, the PLL amines acid group and thus the PLL/amide should be negatively charged were amidized by anhydrides (DCA: 1,2-dicarboxylic-cyclohexene at pH 7.4. As the amides hydrolyze to regenerate the primary anhydride, DM: 2,3-dimethylmaleic anhydride, and TM: 2,2,3,3, amines, the polymer should gradually become positively charged. tetramethylsuccinic anhydride) in water at pH 8.5. The resulting Therefore, the z-potentials of the PLL/amides should change with amides were stable in a weakly basic environment. the hydrolysis of the amides, as shown in Figure 5. At pH 7.4, PLL/ The structures of the polymers were characterized by 1H NMR DM, PLL/DCA, and PLL/TM had a z-potential of about 24, 23, (Fig. 1) and UV (Fig. 2) spectroscopy. The CPT content was and 45 mV, respectively. At pH 7.4 PLL/DM gradually became determined by HPLC using the method reported in the literature.[31] The average number of attached FA molecules was positively charged in 10 h, while PLL/DCA and PLL/TM remained negatively charged even after 24 h. At pH 6.0, the time needed to calculated to be 1.9 per chain from the integration intensities of the become positively charged (z-potential higher than zero) was 0.5 h a-proton in PLL and the aromatic protons in the FA in the NMR for PLL/DM, 7.5 h for PLL/DCA, and 30 h for PLL/TM. At pH 5.0, spectrum, which is in agreement with the results of the UV spectra PLL/DM immediately became positively charged. It took 1.5 h for (2.1 FA groups per chain). The calculated degree of the primary PLL/DCA and 10 h for PLL/TM to become positively charged. amine amidization was higher than 90%. The peaks of the These trends are consistent with the hydrolysis results determined aromatic protons of CPT in the 1H NMR spectra were broad and by 1H NMR spectroscopy in Figure 4. These data also indicate that not clear, probably because of the aggregation of the hydrophobic CPT moieties in water. all the PLL/amides were capable of negative-to-positive charge The content of CPT in FA-PLL/DCA-CPT was determined by reversal. HPLC after it was cleaved from the carrier in the presence of a 10Of the three PLL/amides, PLL/DM had the fastest chargefold molar excess of dithiothreitol (DTT) in a phosphate buffer reversal rates at pH 5.0 and pH 6.0, but it became positively solution (PBS, pH 7.4). The conjugate contained 3.0 wt % of CPT charged at pH 7.4 within only 10 h. This is too fast for in-vivo and less than 0.05 wt % of free CPT. applications because the polymer remaining in the bloodstream for longer than 10 h would become positively charged and thereby 2.2. The pH-Triggered Hydrolysis and Charge Reversal be captured by the reticuloendothelial system rather than tumor tissues. PLL/TM was stable at pH 7.4, but its charge-reversal rate at pH 5 was too slow. PLL/DCA was very stable at pH 7.4 but quickly The carrier, amidized PLL, is expected to be negatively charged at hydrolyzed at pH 6.0 (t1/2 ¼ 6.5 h) and pH 5.0 (t1/2 ¼ 30 min). PLL/ physiological pH (pH 7.4) which inhibits its interaction with cells,


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FULL PAPER Figure 2. The UV spectra of PLL (a), PLL-FITC (b), FA-PLL-FITC (c), and FA-PLL/DCA-CPT (d).

Scheme 3. PLL amidized by DCA (PLL/DCA), DM (PLL/DM), and TM (PLL/TM).

Figure 1. The 1H NMR spectra of PLL/DCA (a), FA-PLL (b), FA-PLL-PDP (c), and FA-PLL/DCA-CPT (d) in D2O.

DCA was always negatively charged at the physiological pH but became positively charged after 6.5 h at pH 6 and fully positively charged within 1.5 h at pH 5.0. Therefore, PLL/DCA is ideal as a charge-reversal carrier for tumor-targeted nuclear drug delivery.

2.3. Cellular Attachment and Uptake At physiological pH, the carrier PLL/DCA is supposed to have very low interaction with cells. In the acidic solid-tumor interstitium, some b-carboxylic acid-amides in the carrier PLL/DCA are

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Figure 3. The hydrolysis of PLL/DM at pH 6.0 detected by 1H NMR spectroscopy.


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Figure 4. The hydrolysis kinetics of PLL/DM, PLL/DCA, and PLL/TM at pH 7.4, 6.0, and 5.0, respectively.

Table 1. The half hydrolysis times (t1/2) of PLL amidized with different anhydrides at different acidities. Polymer

pH 5.0

pH 6.0

pH 7.4

PLL/DM PLL/DCA PLL/TM

60 h >60 h

supposed to hydrolyze and regenerate some primary amines and thus the PLL becomes partially positively charged, which can lead to the conjugate being adsorbed on the negatively charged cellmembrane and trigger the electrostatic adsorptive endocytosis (Scheme 1b, B2). This process was observed by confocal laser scanning fluorescence microscopy and flow cytometry. PLL/DCA was labeled with FITC (PLL/DCA-FITC). PLL/DCA-FITC (zpotential of about 20 mV) was cultured with cells at 37 8C for 15 min and 4 8C for 15 min. No fluorescence was found on the cell membrane (Fig. 6A), which suggests that the negatively charged PLL/DCA did not attach to the negatively charged cell membrane because of electrostatic repulsion. In contrast, if PLL/DCA-FITC was pre-incubated at pH 6 for 6 h, with its increased z-potential of about 5 mV, some fluorescence was found on the cell membrane (Fig. 6B). The cells cultured with PCL/DCA-FITC that had a zpotential of þ5 mVafter further pre-incubation at pH 6 for 24 h had very bright cell membranes, which indicates that the recovered positive charges induced electrostatic adsorption of the PLL/DCAFITC onto the cell membrane (Fig. 6C).

Correspondingly, the cellular uptake of PLL/DCA was measured by flow cytometry (Fig. 7). Cells could not take up untreated PLL/DCA-FITC, but efficiently took it up after it was pretreated for 6 h or 24 h at pH 6 (Fig. 7A). These results further confirm that, once in the acidic extracellular fluid of solid tumor tissues, the PLL/ DCA carrier would be able to become positively charged and thus attach to the cell membranes, and then be quickly taken up (Scheme 1b, B2). The FA targeting groups of the conjugate are also supposed to bind their receptors on the cell membrane and thus trigger receptor-mediated endocytosis. The effectiveness of the targeting group was tested by measuring the cellular uptake of FAfunctionalized PLL/DCA-FITC (FA-PLL/DCA-FITC) using flow cytometry. SKOV-3 ovarian cancer cells overexpress folate receptors[32] and thus were used. As shown in Figure 7B, the cellular uptake of FA-PLL/DCA-FITC was much faster than that of PLL/DCA, which indicates that the FA in FA-PLL/DCA efficiently bound to the receptors on the cell membrane and induced endocytosis (Scheme 1b, B1).

2.4. Subcellular Distribution For nuclear drug delivery, the internalized conjugate must localize in an acidic late endosome/lysosome to regenerate the PLL, and subsequently escape from the lysosome and traverse to the nucleus. A subcellular compartment labeling method was used to observe the subcellular distribution of FA-PLL/DCA-FITC using

Figure 5. The time-dependence of the z-potential as a function of time for PLL/DM, PLL/DCA, PLL/TM (2 mg mL1) in 0.1 M PBS at pH 5.0, 6.0, and 7.4 (n ¼ 3).

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Figure 7. Cellular internalization measured by flow cytometry of A) PLL/DCA-FITC prehydrolyzed at pH 6.0 for 0, 6, and 24 h, respectively, and B) PLL/DCA-FITC and FA-PLL/DCA-FITC. SKOV-3 cells were cultured with 1.4 106 M of the polymers for 12 h. The cell counts were 6000.

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Figure 6. Cell-membrane attachment of PLL/DCA-FITC observed by confocal fluorescence microscopy. PLL/DCA-FITC was prehydrolyzed at pH 6.0 for 0 (A), 6 (B), and 24 h (C). SKOV-3 cells were incubated with the polymer at 37 8C for 15 min and then at 4 8C for 15 min.

confocal microscopy (Figs. 8A–C). The fluorescence of FITC in FA-PLL/DCA-FITC was expressed as green. LysoTracker was used to label late endosomes/lysosomes and displayed as red. The overlay of the two fluorescence images shows yellow spots, which indicates that FA-PLL/DCA-FITC was indeed localized in late endosomes/lysosomes (Scheme 1b, E). The conjugate in a late endosome/lysosome regenerates its positive charges, and it is expected to lyze the lysosomal membrane to escape from it. The lysosomal-lyzing ability of the carrier was estimated using a hemolytic assay of RBCs, a measure of a drug carrier’s ability to rupture lysosomes.[33] RBCs were incubated with four different polymer concentrations in a range of 10–400 mg mL1 (Table 2). Pristine PLL caused 11.6% hemolysis at 10 mg mL1 and 26–30% at higher concentrations in 2 h. PLL/DCA without hydrolysis or hydrolysis for 5 h at pH 6.0 showed no hemolytic ability up to 400 mg mL1. PLL/DCA after being hydrolyzed at pH 6.0 for 24 h showed a hemolytic activity similar to PLL at higher concentrations. The results demonstrate that PLL/DCA will have a similar lysosomal lyzing ability to PLL after regeneration in more acidic lysosomes (pH 4–5) so that the conjugate can escape from the lysosome (Scheme 1b, F). The subsequent nuclear localization of FAPLL/DCA was confirmed by observing the colocalization of FITC-labeled FA-PLL/DCA (FA-PLL/DCA-FITC) and DRAQ-5 labeled nuclei (blue) using confocal microscopy (Figs 8D–F). After a 12 h culture, some FA-PLL-FITC (green) regenerated from FA-PLL/DCA-FITC in the lysosomes was clearly observable in the nuclei, which suggested that the FA-PLL/DCAbased conjugate was capable of accumulating in the nucleus (Scheme 1b, G).

2.5. In Vitro Cytotoxicity

Figure 8. Localization in endosomes/lysosomes (upper panel) and nuclei (lower panel) of FAPLL/DCA-FITC in SKOV-3 cells observed by confocal florescence microscopy. FA-PLL/DCA-FITC (1.4 106 M) was cultured with the cells for 12 h. A) FA-PLL/DCA-FITC, B) LysoTracker, C) overlay of (A) and (B) with transmittance channel, D) FA-PLL/DCA-FITC, E) overlay of (D) with images of nuclear dye and transmittance images, F) and the enlarged image of the selected part in (E).

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The cytotoxicity of free CPT, PLL/DCA, and FAPLL/DCA-CPT to SKOV-3 and MCF-7 cells was evaluated using the (3-(4,5-dimethylthiazolyl2)-2,5-diphenyltetrazolium bromide) (MTT) assay. They were cultured with cells for 24 h, and then the cells were post-cultured for 48 h to allow the damaged cells to undergo apoptosis. The results are presented in Figure 9. PLL is very toxic because of its positive charges, as reported.[34] However, the negatively charged PLL/DCA had a very low cytotoxicity. Its IC50 was higher than 5 mg mL1. The IC50 of the CPT in the FA-PLL/DCA-CPT to SKOV-3 and MCF-7 cells was 3.5 and 0.41 mg mL1, respectively, The FA-PLL/DCA-CPT showed


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Table 2. Hemolysis [mean SD] of sheep RBCs after incubation with different concentrations of the polymers at 37 8C for 120 min [n ¼ 3]. Concentration [mg mL1] 10 50 100 400

PLL/DCA

PLL/DCA [a]

PLL/DCA [b]

PLL

1.42 0.68 1.15 0.36 1.49 0.26 1.83 0.59

3.72 0.23 2.23 0.64 2.60 0.26 3.25 0.38

5.04 0.57 9.43 0.43 15.60 0.91 8.36 0.51

11.64 0.94 28.85 1.59 30.07 0.41 26.00 0.85

[a] Hydrolysis at pH 6 for 5 h. [b] Hydrolysis at pH 6 for 24 h.

obvious dose-dependent cytotoxicity. However, CPT only showed dose dependency at low doses and its cytotoxicity didn’t increase at high doses. This may be a result of the low solubility of CPT in the medium. The enhanced cytotoxicity of CPT in the conjugate may be attributable to its nuclear drug delivery (Scheme 1b, H). CPT binds to and thereby stabilizes topoisomerase I, causing DNA damage and thus cell apoptosis.[35] Thus, free CPT must survive from the various drug resistance mechanisms and traverse to the nucleus before it can elicit its drug action. The CPTconjugated to the carrier is brought to the nucleus by the PLL regenerated in the lysosomes. The disulfide linker can be cleaved by a high concentration of GSH ((0.5–10) 103 M) and thus CPT is released. The CPT released in the nucleus thus circumvents the drug-resistance mechanisms in the cell membrane and cytosol. It should be pointed out that some of the CPT may be released in the cytosol as the conjugate travels to the nucleus because the 3-thiol-propionate-based unhindered disulfide bond has a half life of about 6 h after endocytosis.[36] We are currently solving this problem by optimizing the carrier structure to make the conjugate traverse to the nucleus more quickly and by using more stable disulfide bonds.

3. Conclusions We demonstrate a cancer cell-targeted charge-reversal drug conjugate for nuclear drug delivery to enhance the drug’s cytotoxicity. PLL’s positive charges are masked by converting them into latent amides, which significantly inhibits its ability to interact with cells. Once the amidized PLL is transferred to the cell lysosomes, the amides hydrolyze to regenerate the amines and thus the PLL’s nuclear-localization ability is recovered. By functionalizing the amidized PLL with FA-targeting moieties and an anticancer drug by an intracellular-cleavable disulfide bond, the drug can be efficiently shipped to the cell nucleus to ensure high cytotoxicity.

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4. Experimental

Figure 9. The cytotoxicity of free CPT, PLL/DCA, and FA-PLL/DCA-CPT to SKOV-3 and MCF-7 cells, respectively, estimated by MTT assay. Data represent mean s.d., n ¼ 5.

Materials: DM (98%), DCA (95%), DTT (99þ%), deuterium chloride (35 wt % solution in D2O, 99% D), sodium deuteroxide (40 wt % solution in D2O, 99þ% D), EDC (Commercial Grade), FA (98%), FITC (90þ%), and CPT (95%) were purchased from Sigma–Aldrich and used as received. PLL with a degree of polymerization of about 50 (polydispersity of 1.2) (PLL50),

TM, 3-tritylsulfanylpropionic acid, and SPDP were synthesized according to the reported methods [29,37]. Preparation of FA-PLL (Scheme 2): FA (16.9 mg, 0.038 mmol) was dissolved in anhydrous dimethyl sulfoxide (5 mL) and then EDC (102 mg,


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(PLL/DM, PLL/TM), other amidized products (FA-PLL/DCA), and the charge-reversal conjugate (FA-PLL/DCA-CPT) were prepared similarly. 1H NMR (400 MHz, D2O, d): PLL/DCA: 4.21 (t, 1H), 3.08 (m, 2H), 2.13 (m, 2H), 2.00 (m, 2H), 1.59– 1.67 (m, 4H), 1.51 (m, 4H), 1.28 (m, 2H). PLL/DM: 4.21 (t, 1H), 3.08 (m, 2H), 1.81 (s, 3H), 1.73(s, 3H), 1.59–1.67 (m, 4H), 1.51 (m, 4H), 1.28 (m, 2H). PLL/TM: 4.21 (t, 1H), 3.08 (m, 2H), 1.59–1.67 (m, 4H), 1.51 (m, 4H), 1.28 (m, 2H), 1.07 (s, 6H), 0.98 (s, 6H). FA-PLL/DCA-CPT: 9.0–6.0 (weak, b), 4.21 (t, 1H), 3.08 (m, 2H), 2.13 (m, 2H), 2.00 (m, 2H), 1.59–1.67 (m, 4H), 1.51 (m, 4H), 1.28 (m, 2H). Reverse-phase HPLC: Ion-pairing reverse-phase HPLC (RP-HPLC) was performed on a RP-C18 HPLC column (250 4.6 mm2, 5 micrometers). The mobile phase for elution was 50: 50 water/acetonitrile that contained 0.5% TFA at a flow rate of 1.3 mL min1 for 15 min. The volume of each injection was 100 mL with a sample concentration of approximately 1 mg mL1, and the detection of eluted samples was performed at 360 nm. Amide Hydrolysis Studied by 1H NMR Spectroscopy: The hydrolysis of amidized PLL was monitored by 1H NMR spectroscopy as follows. PLL/ DCA was dissolved in D2O, and the solution pH was adjusted to 7.4, 6.0, or 5.0 using a D2O solution of NaOD or DCl. The solution was warmed in a water bath at 37 8C with shaking. At timed intervals, samples were withdrawn and their 1H NMR spectra were recorded on a Bruker Avance DRX-400 spectrometer. Zeta-Potential Measurements: The z-potentials of the polymers were measured using phase-analysis light-scattering technology. The polymer was dissolved in a PBS (0.1 M) at a pH of 7.4, 6.0, or 5.0 to reach a concentration of 2 mg mL1. The sample was immersed in a water bath at 37 8C with shaking. Samples were taken at timed intervals and their z-potentials were measured using the Zeta-Nanosizer (ZEN3600, Malvern Instruments Ltd, Worcestershire, UK), which was routinely calibrated with a 50 mV zeta-potential standard (Malvern Instruments). The attenuator was set at 9 and the F (Ka) value was set at 1.5. Each measurement was performed for 30 runs, and the results were processed with DTS software version 3.32. Hemolysis Assay: The membrane activity of the polymers was measured using a RBC hemolysis assay [38]. Sheep RBC stock solution was prepared as previously reported [39]. PLL/DCA was prehydrolyzed at pH 6.0 for 0, 5, or 24 h, respectively. The polymers were collected. Each of them was dissolved in PBS at pH 7.4 at concentrations of 0.01, 0.05, 0.1, and 0.4 mg mL1, respectively. The polymer solution (100 mL), gelatin/veronal buffer (GVB, 200 mL), and RBC stock solution (100 mL) were added to a tube. The tube was incubated at 37 8C for 1 h, and then 2 mL of 0.15 M NaCl was added to the tube. The tube was centrifuged (1000g, 3 min) to separate the intact RBCs. The supernatant solution was collected. Absorbance of hemoglobin in the supernatant was measured at 412 nm using a UV-vis spectrophotometer. The observed hemolysis of RBC in PBS (pH 7.4) and in Milli-Q water was used as negative and positive controls, respectively. The observed hemolytic activity of a given polymer at a given concentration value was normalized to that of the positive control, Milli-Q water. All hemolysis experiments were carried out in triplicate. Cell Culture: SKOV-3 adenocarcinoma cells and MCF-7 cells were purchased from American Type Culture Collection (Rockville, MD). Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 10 mg mL1 insulin, and 1% antibiotic/antimycotic solution (Sigma A9909) at 37 8C in a 5% CO2 environment. The cells used for cellular uptake and in-vitro cytotoxicity assay of the FA-functionalized PLL or conjugate were cultured in folic-free medium (Invitrogen Corp.) for at least two weeks before use. Polymer Adsorption on the Cell Membrane: Polymer adsorption on the cell membrane was observed by confocal fluorescence microscopy: Cells were seeded onto 35 mm optical dishes (MatTek) at 80 000 cells per plate in 2 mL of medium and incubated overnight. PLL/DCA-FITC was prehydrolyzed at pH 6.0 for 0, 5, or 24 h. FA-PLL/DCA-FITC was used directly. One milliliter of the cell culture medium was replaced with 1 mL of fresh medium that contained 1.4 106 M polymer. The cells were incubated at 37 8C and 5% CO2 for 1 h. Cells were then washed with cold


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0.53 mmol) was added to this solution while stirring in the dark at room temperature for 1 h. The solution was added to the PLL50 (100 mg, 0.48 mmol NH2 groups) solution in 20 mL of water at pH 8.5. The solution was stirred for 2 days in the dark. The mixture was dialyzed (MWCO ¼ 3500 Da) against water (2 L 4) for 2 days, and was lyophilized with a yield of 91%. The product was confirmed by 1H NMR spectroscopy and the folate content was determined by UV (e363 ¼ 6200) spectroscopy. 1 H NMR (400 MHz, D2O, d): 8.57 (s, 1H), 7.57 (d, 2H), 6.67 (d, 2H), 4.23 (t, 1H), 2.91 (t, 2H), 1.60–1.81 (m, 4H), 1.32 (m, 2H). Labeling PLL or FA-PLL with FITC: PLL or FA-PLL (100 mg, 0.48 mmol NH2 groups) was dissolved in 10 mL of water and the pH was adjusted to 9.4. FITC (10 mg) was added to this solution, and the solution was stirred overnight in the dark. The solution was then dialyzed (Spectra/Pro MWCO ¼ 3500 Da) against water to remove the unreacted FITC. Finally, the purified solution was lyophilized to obtain a yellow solid with a yield of 85%. 1H NMR (400 MHz, D2O, d): 7.0–7.8 (br, 3H), 6.3–6.6 (br, 5H), 4.22 (t, 1H), 2.91 (m, 2H), 1.63–1.79 (m, 4H), 1.30 (m, 2H). Preparation of CPT-SH (Scheme 2): CPT (348 mg, 1.0 mmol), 3-tritylsulfanylpropionic acid (348 mg, 1.0 mmol), 4-dimethylaminopyridine (DMAP, 134 mg, 1.1 mmol) and EDC (210 mg, 1.1 mmol) were mixed in anhydrous dichloromethane (50 mL). The solution was stirred overnight at room temperature. The solution was then filtered and the solvent was removed by rotary evaporation. The residue was dissolved in 14 mL of anhydrous dichloromethane, and triethylsilane (2 mL) was added followed by trifluoroacetic acid (4 mL). After 2 h stirring at room temperature, dichloromethane and trifluoroacetic acid were removed by evaporation. The crude product was redissolved in dichloromethane and the solution was poured into an excess of cold ethyl ether. The precipitate was isolated and dried under vacuum. A yellowish powder with a purity of 92% (according to 1H NMR spectroscopy) was obtained with a yield of 80%. 1H NMR (400 MHz, DMSO-d6, d): 8.68 (s 1H), 8.15 (m, 2H), 7.86 (m, 1H), 7.71 (m, 1H), 7.17(s, 1H), 5.51 (s, 2H), 5.29 (s, 2H), 3.01 (m, 1H), 2.92 (t, 2H), 2.73 (m, 2H) 2.17 (m, 2H), 0.93 (t, 3H). Preparation of the FA-PLL-CPT Conjugate (Scheme 2): CPT was introduced to FA-PLL by a disulfide bond using a heterobifunctional reagent, SPDP, and the CPT-SH. A typical synthetic procedure is as follows. FA-PLL (100 mg, 0.78 mmol NH2 groups) was dissolved in water (10 mL). SPDP (10 mg, 0.032 mmol) in methanol (1.3 mL) was dropped into the solution. The reaction was kept in the dark for 2 h, and then the solution was dialyzed (Spectra/Pro MWCO ¼ 1000) against water. The solution was dried to give 90 mg of FA-PLL-PDP. The degree of substitution was determined from the peak intensity ratio of the methylene protons of PLL (CH2NH2, d ¼ 2.91 ppm) and the pyridyl protons (C5H4N, d ¼ 7.2– 8.3 ppm) of the pyridyldithiopropionyl (PDP) groups in the 1H NMR spectrum and the absorbance of 2-thiopyridone (lmax ¼ 343 nm; e ¼ 7.06 103) released after the addition of DTT. The obtained FA-PLLPDP (6.7 mol % substitution; 90 mg, 0.042 mmol PDP) was redissolved in a mixed solvent of N,N-dimethylformamide (DMF, 10 mL) and water (4 mL). CPT-SH (34 mg, 0.084 mmol) was added to the solution and stirred for 2 h. De-ionized water (15 mL) was added to the solution to precipitate out the unreacted CPT. After filtration, the solution was dialyzed (Spectra/Pro MWCO ¼ 3500) against acetonitrile (200 mL 3) and water (2 L 3). Finally, the solution was lyophilized to give 50 mg of a yellow solid. The CPT content was determined by HPLC. 1H NMR for FA-PLL-PDP (400 MHz, D2O, d): 8.57 (s 1H), 8.29 (m, 1H), 7.69 (m, 2H), 7.57 (d, 2H), 7.18 (m, 1H), 6.67 (d, 2H), 4.21 (t, 1H), 2.91 (m, 2H), 2.52 (t, 2H), 1.60–1.81 (m, 4H), 1.32 (m, 2H). 1H NMR for FA-PLL-PDP (400 MHz, D2O, d): 9.0–6.0 (b), 4.21 (t, 1H), 2.91 (m, 2H), 2.52 (t, 2H), 1.60–1.81 (m, 4H). Preparation of PLL/DCA, FA-PLL/DCA, and FA-PLL/DCA-FITC and the Conjugate FA-PLL/DCA-CPT (Scheme 2): A typical procedure to amidize PLL or functionalized PLL (FA-PLL and FA-PLL-FITC) using anhydrides is as follows: PLL (100 mg, 0.78 mmol NH2 groups) was dissolved in 2 mL of deionized water and the solution pH was adjusted to 8.5. DCA (1.2 g, 7.8 mmol) was gradually added to the solution and the pH was kept at 8.5 by adding 3 N NaOH during the reaction. The reaction was allowed to continue for an additional half hour after the DCA was completely added. The solution was dialyzed against water at pH 8.5. The product (PLL/DCA) was obtained after freeze drying. PLL amidized by the other anhydrides

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PBS (4 8C) three times. One milliliter of cold PBS was then added to the dish and the cells were observed with a confocal laser light scanning microscope (excitation 488 nm and emission 505–530 nm). Cells were kept at 4 8C except when being observed under the microscope. To test the accumulation of free dye in the cells from the solution, 1.6 mL of the polymer solution was filtered through a Centricon filter with a 3000-molecular-weight cutoff (Amicon, Bedford, MA, no. YM-3) at 2600g for 1 h. The free dye passed through the filter (filtrate) while the polymer was retained on the filter. The filtrate was then applied to the cells and the cells were observed under identical conditions. Cellular Uptake and Intracellular Colocalization of the Polymers and Conjugate: Cellular uptake and intracellular colocalization of the polymers and conjugate was observed using confocal fluorescence microscopy. Cells were incubated with 1.4 106 M of each polymer at 37 8C and 5% CO2 for 10 h. LysoTracker Red DND-99 (Molecular Probes, Eugene, OR) (150 109 M) was then added and the cells were incubated for 2 h. The cells were then thoroughly washed with PBS at 4 8C three times. DRAQ 5 nuclei dye (1 mL) (Biostatus, 5 103 M in dilute acid) was added just before observation. Images were obtained using a Leica TCS SP2 microscope. LysoTracker was observed using a GreNe 543 nm laser and the emission wavelength was read from 523 to 563 nm and expressed as red. DRAQ 5 nuclei dye was observed using a HeNe 633 nm laser and the emission wavelength was read from 660 to 760 nm and expressed as blue. Polymers labeled with FITC were observed using a Ar/ArKr 458/488 nm laser and the emission wavelength was read from 505 to 530 nm and expressed as green. To test the accumulation of free dye in the cells from the polymer solution, 1.6 mL of the polymer solution was filtered through a Centricon filter with a 3000-molecular-weight cutoff (Amicon, Bedford, MA, no. YM-3) at 2600g for 1 h. Unconjugated dye passed through the filter and into the filtrate while polymer-conjugated FITC was retained on the filter. The filtrate was then applied to the cells and the cells were observed similarly. Cellular Uptake Measured by Flow Cytometry: Cells were seeded onto six-well plates (2.5 mL of cell suspension per well) at 2 105 cells mL1 and allowed to grow for 24 h. The polymers (1.4 106 M) were added to each well and incubated with cells for 12 h. The cells were then washed with cold PBS twice, harvested by 0.25% (w/v) trypsin/0.03% (w/v) ethylenediaminetetraacetic acid (EDTA), pelleted in eppendorfs and centrifuged at 1000g for 4 min at 4 8C, and then resuspended in PBS with 2% FBS. Each sample was quickly analyzed on a NPE QUANTA (NPE SYSTEMS, Pembroke Pines, FL) using the 488 nm argon laser for excitation and the emitted 525 nm fluorescence for detection. Files were collected of 6000 gated events and analyzed with the NPE Quanta 2.1 software program. In-vitro Cytotoxicity Assay: The cytotoxicity assay was carried out using the MTT cell proliferation assay kit (ATCC, Manassas, VA) according to the modified manufacturer’s protocol. SKOV-3 and MCF-7 cells were cultured in folic-free medium (Invitrogen Corp., Carlsbad, CA) for at least two weeks before use. They were then seeded onto 96-well plates at a density of 10 000 cells per well and incubated for 24 h. The original medium (200 mL) was removed and replaced with the polymer, conjugate, or free CPT solutions at different concentrations and incubated for 24 h. The medium in each well was then replaced with fresh cell culture medium and further incubated for 48 h. MTT reagent (10 mL) was then added to each well and incubated for 6 h until purple precipitates were visible. Finally, the detergent reagent (100 mL) was added to each well, and the plates were incubated at 37 8C for 18 h until all the crystals were dissolved. The absorbance intensity at 570 nm was recorded and the cytotoxicity was expressed as a percentage of the control.

Acknowledgements The authors thank the National Basic Research Program (973 Program, 2009CB526403) of China, National Fund for Distinguished Young Scholars of China (50888001), and the US Department of Defense (BC062422) for financial support. Received: May 12, 2009 Published online: September 24, 2009

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