Highly Efficient Nanomedicines Assembled Via Polymer-drug Multiple Interactions

The clinical applications of many important therapeutics are restricted due to their poor water solubility. Accordingly, it is highly necessary to develop effective delivery systems to facilitate their clinic use. As an ideal drug delivery system, it must be able to transport drugs to specific sites of the body and release the payload in the targets of interest. Meanwhile, the carrier itself must be nontoxic or it can be degraded into nontoxic byproducts in the body. In recent years, much attention has been paid to novel drug delivery systems based on polymer assemblies constructed by macromolecular amphiphiles. These novel carriers have been widely studied for the delivery of a broad spectrum of therapeutics varying from small molecular drugs, proteins, and genes, mainly due to their unique features such as colloidal stablility and potential targetability.Recent progress in synthetic chemistry has dramatically promoted the design and preparation of intelligent polymeric micelles that can specifically target diseased sites and release the cargo molecules under the external stimuli. Currently, there are more than six formulations based on polymer nanoassemblies are under clinical trials, which have been developed as delivery carriers of anti-cancer drugs such as doxorubicin, paclitaxel, and camptothecin. Nevertheless, nanomedicines based on polymer assemblies frequently encounter the inherent drawback of low drug loading capacity for hydrophobic drugs, which may restrict the therapeutic efficacy in the final therapy stage or even influence the normal efficacy of delivered drugs. For most nanocarriers assembled via molecular amphiphiles, drug molecules are passively incorporated via hydrophobic interaction. This single interaction between polymer and drug is mainly responsible for the low drug loading content. To circumvent this issue, we hypothesize that multiple nonconvalent interactions between polymer and drug may enhance the encapsulation of drug molecules into nanoassemblies. As a proof of concept, we select indomethacin (IND) as a model drug. To reach the polymer-drug multiple interactions,β-cyclodextrin (β-CD) conjugated polyethyleneimine (PEI-CD) was synthesized. Since PEI-CD contains primary, secondary, and tertiary amines as well asβ-CD units, there should be electrostatic, hydrogen-bonding, and inclusion interactions between PEI-CD and IND. In addition, the hydrophobic interactions of drug molecules may also facilitate the construction of highly efficient nanomedicines.PEI-CD was synthesized by a nucleophilic substitution reaction between branched polyethyleneimine (PEI) and 6-monotosylβ-CD, and its structure was characterized by 1H-NMR and FT-IR. The molar ratio of ethyleneimine units in PEI toβ-CD groups was calculated according to 1H-NMR spectrum.Highly efficient delivery vehicles that can release drugs in enteric sites post oral administration were then constructed based on the multiple interactions of PEI-CD and IND. The involved noncovalent forces include electrostatic, hydrogen-bonding, host-guest recognition and hydrophobic interactions. IND/PEI-CD nanoassemblies were prepared via modified dialysis procedure. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were adopted to observe the morphology of assemblies formulated with various drug/polymer ratios. Noncovalent interactions between polymer and drug were characterized by FT-IR and NMR. The existence form of drug molecules in assemblies was determined by differential scanning calorimetry (DSC), x-ray diffraction (XRD), and confocal laser scanning microscopy (CLSM). Drug loading and entrapment efficiency of IND/PEI-CD nanoassemblies were quantified by UV, suggesting that the highest IND content can be up to 71%. High drug loading capacity was also achieved for other carboxyl-containing drugs, including ibuprofen, diflunisal, flurbiprofen, and naproxen, with the drug loading content higher than 50%. Copolymers of ethylene oxide (EO)/propylene oxide (PO) and PEG5000-b-PLLA5000 were used for comparison. The highest IND loading achieved was 22.9%, and increasing IND feed could not further enhance drug content in the resultant assemblies. Assemblies based on PEG5000-b-PLLA5000 containing IND was also fabricated, and the highest drug loading for this system was 18.9%. In vitro drug release tests were performed in 0.01 M PBS (pH 7.4) as well as in solutions simulating gastrointestinal (GI) conditions. It was found that drug in IND/PEI-CD can be rapidly released in PBS of pH 7.4, with a cumulative release percentage of 90% within 24 h. In the case of release simulating the GI tract conditions, almost no release was observed in simulated gastric solution, while > 90% of total amount could be released in simulated enteric buffer. In contrast, only 22% was released for raw IND under the same conditions. Preliminary pharmacokinetic studies in rats revealed that relatively high drug concentration could be maintained for up to 4 days after oral administration of IND/PEI-CD nanoassemblies at a dose of 10 mg/kg. The area under concentration-time curve (AUC) was 9 times higher than that of raw IND, suggesting that the oral bioavailability of IND isdramatically enhanced by formulating into nanomedicines via molecular self-assembly. Tracing using fluorescence microscopy based on fluorescence-labeled PEI-CD indicated that IND/PEI-CD nanoassemblies could retained in GI tract for up to 36 h, which may contribute for the sustained drug levels in plasma post oral administration.Methods1. Sythesis ofβ-cyclodextrin-conjugated polyethyleneimine (PEI-CD) PEI-CD was synthesized by a nucleophilic substitution reaction between branched PEI and 6-monotosylβ-CD.2. Polymer characterization The structure of PEI-CD was characterized by 1H-NMR and FT-IR. The molar ratio of ethyleneimine units in PEI toβ-CD groups was calculated via 1H-NMR spectrum.3. Fabrication of drug/PEI-CD nanoassemblies A modified dialysis process was used to prepare drug/PEICD assemblies. Briefly, an appropriate amount of drug dissolved in DMSO was gradually added into an aqueous solution of PEI-CD (10.0 mg/mL) under bath sonication. The obtained mixture was then dialyzed against deionized water to form drug-containing nanoassemblies. The involved drugs include IND, naproxen, ibuprofen, flurbiprofen, and diflunisal.4. Quantification of drug content in assemblies An appropriate amount of lyophilized drug-loaded nanoassemblies was dispersed in ethanol that is a good solvent for all the employed drugs, while a nonsolvent for PEI-CD. Drug in the lyophilized samples was extracted into ethanol by three times of extraction, and then drug concentration in ethanol was determined at 310, 306, 273, 247, and 271 nm for IND, diflunisal, ibuprofen, flurbiprofen, and naproxen, respectively. Drug loading content and entrapment efficiency were calculated according to the following formulas. Loading content (%) = TheT wheei gwheti ogfh td oruf gn ainn onaasnsoeamssbelimesb l(imesg ()m g)×100% Entrapment efficiency (%) = Drug content in nanoassemblies (%) 100%Theoretical drug content (%)×5. Characterization of multiple interactions between polymer and drug Raw IND, IND/PEI-CD physical mixture, and IND/PEI-CD assemblies containing various contents of IND were examined by FT-IR, 1H and 1H-1H Roesy NMR measurements.6. Morphology observation and zeta-potential measurement Morphology observation of drug-containing nanoassemblies was performed by TEM, SEM, and atomic force microscopy (AFM).7. Characterization of the drug form in assemblies Raw IND, IND/PEI-CD physical mixture and IND/PEI-CD assemblies containing various contents of IND were examined by DSC and XRD measurement.8. Decoration of IND/PEI-CD assemblies DMSO solution containing IND was added into aqueous solution containing both PEI-CD and PEG-Ada under bath soniction, and the obtained solution mixture was dialyzed against deionized water for 24 h to obtain peripherally PEGylated nanoassemblies.9. In vitro release test 0.5 mL of IND/PEI-CD assemblies was placed into dialysis tubing, which was immerged into 40 mL of PBS (pH 7.4). At predetermined time intervals, 4.0 mL of release medium was withdrawn, and fresh PBS was added. To simulate release profiles under GI tract conditions, aqueous solution of HCl (pH 1.2) was employed within the first two hours, and then the release medium was switched into 0.01 M PBS (pH 7.4). IND concentration in release buffer was quantified by UV at 310 nm.10. Pharmacokinetic study IND/PEI-CD assemblies and raw IND were orally administered via gastric gavage. Blood samples were collected at specific time points post-dose. Plasma was obtained after 10 min of centrifugation at 3000 rpm. Then, 100 mL of acetonitrile and 400 mL of acetonitrile was added into 50μL plasma, followed by votexing for 1 min, and the supernatant was withdrawn after centrifugation at 8000 rpm for 10 min. After being dried under N2 atmosphere, 100μL of mobile phase was added for sampling. IND concentration in was quantified by high performance liquid chromatography. The chromatographic conditions are as follows: mobile phase, acetonitrile-6μM H3PO4 (55:45, v:v); eluent rate, 1.0 mL/min; column temperature, 40°C; detection wavelength, 245 nm; sample volume, 20μL; column, C18 reverse column (5μm×250 mm).11. Study on the retention and GI irritation IND/PEI-CD assemblies were orally administered to SD male rats at 10 mg/kg. At predetermined time points, they were euthanized and isolated segments of stomach and small intestine tissues were fixed. Histological sections were made and stained with hematoxylin-eosin (HE). Using the fluorescence-labeled PEI-CD, the distribution of IND-containing assemblies in intestinal tract was detected at various time points.Results1. Copolymer ofβ-CD conjugated PEI (PEI-CD) that contains primary, secondary, and tertiary amines as well asβ-CD units was successfully synthesized. According to 1H-NMR spectrum, the molar ratio of ethyleneimine units toβ-CD groups is about 15.2:1 for the newly synthesized PEI-CD.2. Nanoassemblies were constructed taking advantage of the multiple interactions of drug/PEI-CD. For IND assemblies, they exhibited drug loading content higher than 71% and encapsulation efficiency > 90%, while it can be higher than 50% for other carboxyl-containing drugs like naproxen, diflunisal, ibuprofen, and flurbiprofen.3. Measurements based on FT-IR, 1H and 1H-1H Roesy NMR confirmed the presence of non-covalent multiple interactions between IND/PEI-CD, including hydropobic, host-guest recognition, hydrogen-bonding and electrostatic forces.4. Morphology observation combined with size determination revealed thus obtained nanoassemblies displayed spherical shape with mean size less than 200 nm. In addition, these drug-containing assemblies are positively charged according to the zeta-potential measurement.5. DSC and XRD measurements as well as CLSM observation indicated the IND molecules encapsulated in nanoparticles were essentially amorphous other than drug crystal.6. PEGylation of IND/PEI-CD nanoassemblies can be successfully performed using PEG-Ada, and the obtained nanoparticles are sphere in shape with good dispersibility. 7. Fast release of IND was achieved at pH 7.4. In vitro release in media simulating GI conditions suggested that the drug release was largely suppressed in acidic conditions, which can be dramatically accelerated when the release medium was switched into release buffer of pH 7.4. In addition, the release of IND was faster than that of raw IND and commercial tablet.8. Pharmacokinetic study was performed in SD rats after oral administration. The relatively high level of IND was maintained up to 4 days in the case of nano-assemblies. The area under the plasma concentration-time curve (AUC) of nanomedicines was about 9-fold larger than that of raw IND.9. Observation using fluorescence microscopy indicated that IND/PEI-CD nanoassemblies can be retained in GI tract for up to 36 h. Pathological study showed slight irritation on stomach 0.5 and 1 h post-gavage.Conclusions1. Through a nucleophilic reaction between mono-6-tosylβ-CD and PEI, hydrophilic copolymer PEI-CD that contains primary, secondary, and tertiary amines as well asβ-CD units was successfully synthesized.2. Based on the multiple noncovalent interactions between carboxyl-containing drugs and PEI-CD, core-shell structured nanoassemblies were prepared by a modified dialysis. Compared with polymer micelles assembled via hydrophobic interactions alone, the newly developed nanoparticles displayed higher drug loading capacity. Therefore, the aforementioned hypothesis that high drug loaded nanoassemblies can be constructed using polymers possessing multiple interactions with drug molecules, was solidly confirmed.3. The colloidal stability and redispersibility of IND/PEI-CD assemblies can be significantly enhanced through PEGylation using PEG-Ada. Additionally, these nanocarriers can be used to deliver other hydrophobic drugs.4. Drug molecules in these nanomedicines can be rapidly released at pH 7.4, and therefore they can enhance the drug dissolution. The drug oral bioavailability can be dramatically enhanced by formulating into nanomedicines via molecular self-assembly. The nanomedicines displayed significantly reduced stomach irritation in comparison to raw IND, and assemblies can be maintained in enteric sites for a long time after oral administration. IND/PEI-CD nanoassemblies are positively charged, these nanoparticles with positive peripheral may serve as carriers for the transport of some biomacromolecules, such as genetic therapeutics.