Structure and Dynamics of Thermosensitive pDNA Polyplexes Studied by Time-Resolved Fluorescence Spectroscopy

Combining multiple stimuli-responsive functionalities into the polymer design is an attractive approach to improve nucleic acid delivery. However, more in-depth fundamental understanding how the multiple functionalities in the polymer structures are influencing polyplex formation and stability is essential for the rational development of such delivery systems. Therefore, in this study the structure and dynamics of thermosensitive polyplexes were investigated by tracking the behavior of labeled plasmid DNA (pDNA) and polymer with time-resolved fluorescence spectroscopy using fluorescence resonance energy transfer (FRET). The successful synthesis of a heterofunctional poly(ethylene glycol) (PEG) macroinitiator containing both an atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain-transfer (RAFT) initiator is reported. The use of this novel PEG macroinitiator allows for the controlled polymerization of cationic and thermosensitive linear triblock copolymers and labeling of the chain-end with a fluorescent dye by maleimide-thiol chemistry. The polymers consisted of a thermosensitive poly(N-isopropylacrylamide) (PNIPAM, N), hydrophilic PEG (P), and cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA, D) block, further referred to as NPD. Polymer block D chain-ends were labeled with Cy3, while pDNA was labeled with FITC. The thermosensitive NPD polymers were used to prepare pDNA polyplexes, and the effect of the N/P charge ratio, temperature, and composition of the triblock copolymer on the polyplex properties were investigated, taking nonthermosensitive PD polymers as the control. FRET was observed both at 4 and 37 degrees C, indicating that the introduction of the thermosensitive PNIPAM block did not compromise the polyplex structure even above the polymer's cloud point. Furthermore, FRET results showed that the NPD- and PD-based polyplexes have a less dense core compared to polyplexes based on cationic homopolymers (such as PEI) as reported before. The polyplexes showed to have a dynamic character meaning that the polymer chains can exchange between the polyplex core and shell. Mobility of the polymers allow their uniform redistribution within the polyplex and this feature has been reported to be favorable in the context of pDNA release and subsequent improved transfection efficiency, compared to nondynamic formulations.Peer reviewe

The in-vitro effect of complementary and alternative medicines on cytochrome P450 2C9 activity

OBJECTIVES: The aim of this study is to establish the inhibitory effects of 14 commonly used complementary and alternative medicines (CAM) on the metabolism of cytochrome P450 2C9 (CYP2C9) substrates 7-methoxy-4-trifluoromethyl coumarine (MFC) and tolbutamide. CYP2C9 is important for the metabolism of numerous drugs and inhibition of this enzyme by CAM could result in elevated plasma levels of drugs that are CYP2C9 substrates. Especially for anticancer drugs, which have a narrow therapeutic window, small changes in their plasma levels could easily result in clinically relevant toxicities. METHODS: The effects of CAM on CYP2C9-mediated metabolism of MFC were assessed in Supersomes, using the fluorometric CYP2C9 inhibition assay. In human liver microsomes (HLM) the inhibition of CYP2C9-mediated metabolism of tolbutamide was determined, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). KEY FINDINGS: The results indicated milk thistle as the most potent CYP2C9 inhibitor. For milk thistle, silybin (main constituent of milk thistle) was mainly responsible for the inhibition of CY2C9. CONCLUSIONS: Milk thistle and green tea were confirmed as potent inhibitors of CYP2C9-mediated metabolism of multiple substrates in vitro. Clinical studies with milk thistle are recommended to establish the clinical relevance of the demonstrated CYP2C9 inhibition

The in-vitro effect of complementary and alternative medicines on cytochrome P450 2C9 activity

OBJECTIVES: The aim of this study is to establish the inhibitory effects of 14 commonly used complementary and alternative medicines (CAM) on the metabolism of cytochrome P450 2C9 (CYP2C9) substrates 7-methoxy-4-trifluoromethyl coumarine (MFC) and tolbutamide. CYP2C9 is important for the metabolism of numerous drugs and inhibition of this enzyme by CAM could result in elevated plasma levels of drugs that are CYP2C9 substrates. Especially for anticancer drugs, which have a narrow therapeutic window, small changes in their plasma levels could easily result in clinically relevant toxicities. METHODS: The effects of CAM on CYP2C9-mediated metabolism of MFC were assessed in Supersomes, using the fluorometric CYP2C9 inhibition assay. In human liver microsomes (HLM) the inhibition of CYP2C9-mediated metabolism of tolbutamide was determined, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). KEY FINDINGS: The results indicated milk thistle as the most potent CYP2C9 inhibitor. For milk thistle, silybin (main constituent of milk thistle) was mainly responsible for the inhibition of CY2C9. CONCLUSIONS: Milk thistle and green tea were confirmed as potent inhibitors of CYP2C9-mediated metabolism of multiple substrates in vitro. Clinical studies with milk thistle are recommended to establish the clinical relevance of the demonstrated CYP2C9 inhibition

The effect of complementary and alternative medicines on CYP3A4-mediated metabolism of three different substrates : 7-benzyloxy-4-trifluoromethyl-coumarin, midazolam and docetaxel

OBJECTIVE: Concomitant use of complementary and alternative medicine (CAM) and anticancer drugs can affect the pharmacokinetics of anticancer drugs by inhibiting the metabolizing enzyme cytochrome P450 3A4 (CYP3A4) (EC 1.14.13.157). Several in vitro studies determined whether CAM can inhibit CYP3A4, but these studies revealed contradictory results. A plausible explanation for these conflicting results is the use only of a single model CYP3A4 substrate in each study. Therefore, the objective was to determine the potential of selected CAM (β-carotene, Echinacea, garlic, Ginkgo biloba, ginseng, grape seed extract, green tea extract, milk thistle, saw palmetto, valerian, vitamin B6, B12 and C) to inhibit CYP3A4-mediated metabolism of different substrates: 7-benzyloxy-4-trifluoromethyl-coumarin (BFC), midazolam and docetaxel. The effect of CAM on CYP3A4-mediated metabolism of an anticancer drug has never been determined before in vitro, which makes this study unique. The oncolytic CYP3A4 substrate docetaxel was used to establish the predictive value of the model substrates for pharmacokinetic interactions between CAM and anticancer drugs in vitro, and to more closely predict these interactions in vivo. METHODS: The inhibition of CYP3A4-mediated metabolism of 7-benzyloxy-4-trifluoromethyl-coumarin (BFC) by CAM was assessed in Supersomes, using the fluorometric CYP3A4 inhibition assay. In human liver microsomes (HLM) the inhibition of CYP3A4-mediated metabolism of midazolam and docetaxel was determined, using liquid-chromatography coupled to tandem mass spectrometry (LC-MS/MS). KEY FINDINGS: The results confirmed grape seed and green tea as potent inhibitors and milk thistle as moderate inhibitor of CYP3A4-mediated metabolism of BFC, midazolam and docetaxel. CONCLUSION: Clinical studies are required to determine the clinical relevance of the determined CYP3A4 inhibition by grape seed, green tea and milk thistle

The effect of complementary and alternative medicines on CYP3A4-mediated metabolism of three different substrates : 7-benzyloxy-4-trifluoromethyl-coumarin, midazolam and docetaxel

OBJECTIVE: Concomitant use of complementary and alternative medicine (CAM) and anticancer drugs can affect the pharmacokinetics of anticancer drugs by inhibiting the metabolizing enzyme cytochrome P450 3A4 (CYP3A4) (EC 1.14.13.157). Several in vitro studies determined whether CAM can inhibit CYP3A4, but these studies revealed contradictory results. A plausible explanation for these conflicting results is the use only of a single model CYP3A4 substrate in each study. Therefore, the objective was to determine the potential of selected CAM (β-carotene, Echinacea, garlic, Ginkgo biloba, ginseng, grape seed extract, green tea extract, milk thistle, saw palmetto, valerian, vitamin B6, B12 and C) to inhibit CYP3A4-mediated metabolism of different substrates: 7-benzyloxy-4-trifluoromethyl-coumarin (BFC), midazolam and docetaxel. The effect of CAM on CYP3A4-mediated metabolism of an anticancer drug has never been determined before in vitro, which makes this study unique. The oncolytic CYP3A4 substrate docetaxel was used to establish the predictive value of the model substrates for pharmacokinetic interactions between CAM and anticancer drugs in vitro, and to more closely predict these interactions in vivo. METHODS: The inhibition of CYP3A4-mediated metabolism of 7-benzyloxy-4-trifluoromethyl-coumarin (BFC) by CAM was assessed in Supersomes, using the fluorometric CYP3A4 inhibition assay. In human liver microsomes (HLM) the inhibition of CYP3A4-mediated metabolism of midazolam and docetaxel was determined, using liquid-chromatography coupled to tandem mass spectrometry (LC-MS/MS). KEY FINDINGS: The results confirmed grape seed and green tea as potent inhibitors and milk thistle as moderate inhibitor of CYP3A4-mediated metabolism of BFC, midazolam and docetaxel. CONCLUSION: Clinical studies are required to determine the clinical relevance of the determined CYP3A4 inhibition by grape seed, green tea and milk thistle

π-π-Stacked Poly(ε-caprolactone)-b-poly(ethylene glycol) Micelles Loaded with a Photosensitizer for Photodynamic Therapy

To improve the in vivo stability of poly(-caprolactone)-b-poly(ethylene glycol) (PCL-PEG)-based micelles and cargo retention by π-π stacking interactions, pendant aromatic rings were introduced by copolymerization of -caprolactone with benzyl 5-methyl-2-oxo-1,3-dioxane-5-carboxylate (TMC-Bz). It was shown that the incorporation of aromatic rings yielded smaller micelles (18-30 nm) with better colloidal stability in PBS than micelles without aromatic groups. The circulation time of i.v. injected micelles containing multiple pendant aromatic groups was longer (t½-α: ~0.7 h; t½-β: 2.9 h) than that of micelles with a single terminal aromatic group (t½ < 0.3 h). In addition, the in vitro partitioning of the encapsulated photosensitizer (meta-tetra(hydroxyphenyl)chlorin, mTHPC) between micelles and human plasma was favored towards micelles for those that contained the pendant aromatic groups. However, this was not sufficient to fully retain mTHPC in the micelles in vivo, as indicated by similar biodistribution patterns of micellar mTHPC compared to free mTHPC, and unequal biodistribution patterns of mTHPC and the host micelles. Our study points out that more detailed in vitro methods are necessary to more reliably predict in vivo outcomes. Furthermore, additional measures beyond π-π stacking are needed to stably incorporate mTHPC in micelles in order to benefit from the use of micelles as targeted delivery systems

π-π-Stacked Poly(ε-caprolactone)-b-poly(ethylene glycol) Micelles Loaded with a Photosensitizer for Photodynamic Therapy

To improve the in vivo stability of poly(-caprolactone)-b-poly(ethylene glycol) (PCL-PEG)-based micelles and cargo retention by π-π stacking interactions, pendant aromatic rings were introduced by copolymerization of -caprolactone with benzyl 5-methyl-2-oxo-1,3-dioxane-5-carboxylate (TMC-Bz). It was shown that the incorporation of aromatic rings yielded smaller micelles (18-30 nm) with better colloidal stability in PBS than micelles without aromatic groups. The circulation time of i.v. injected micelles containing multiple pendant aromatic groups was longer (t½-α: ~0.7 h; t½-β: 2.9 h) than that of micelles with a single terminal aromatic group (t½ < 0.3 h). In addition, the in vitro partitioning of the encapsulated photosensitizer (meta-tetra(hydroxyphenyl)chlorin, mTHPC) between micelles and human plasma was favored towards micelles for those that contained the pendant aromatic groups. However, this was not sufficient to fully retain mTHPC in the micelles in vivo, as indicated by similar biodistribution patterns of micellar mTHPC compared to free mTHPC, and unequal biodistribution patterns of mTHPC and the host micelles. Our study points out that more detailed in vitro methods are necessary to more reliably predict in vivo outcomes. Furthermore, additional measures beyond π-π stacking are needed to stably incorporate mTHPC in micelles in order to benefit from the use of micelles as targeted delivery systems

Structure and Dynamics of Thermosensitive pDNA Polyplexes Studied by Time-Resolved Fluorescence Spectroscopy

Combining multiple stimuli-responsive functionalities into the polymer design is an attractive approach to improve nucleic acid delivery. However, more in-depth fundamental understanding how the multiple functionalities in the polymer structures are influencing polyplex formation and stability is essential for the rational development of such delivery systems. Therefore, in this study the structure and dynamics of thermosensitive polyplexes were investigated by tracking the behavior of labeled plasmid DNA (pDNA) and polymer with time-resolved fluorescence spectroscopy using fluorescence resonance energy transfer (FRET). The successful synthesis of a heterofunctional poly(ethylene glycol) (PEG) macroinitiator containing both an atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain-transfer (RAFT) initiator is reported. The use of this novel PEG macroinitiator allows for the controlled polymerization of cationic and thermosensitive linear triblock copolymers and labeling of the chain-end with a fluorescent dye by maleimide-thiol chemistry. The polymers consisted of a thermosensitive poly(N-isopropylacrylamide) (PNIPAM, N), hydrophilic PEG (P), and cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA, D) block, further referred to as NPD. Polymer block D chain-ends were labeled with Cy3, while pDNA was labeled with FITC. The thermosensitive NPD polymers were used to prepare pDNA polyplexes, and the effect of the N/P charge ratio, temperature, and composition of the triblock copolymer on the polyplex properties were investigated, taking nonthermosensitive PD polymers as the control. FRET was observed both at 4 and 37 °C, indicating that the introduction of the thermosensitive PNIPAM block did not compromise the polyplex structure even above the polymer's cloud point. Furthermore, FRET results showed that the NPD- and PD-based polyplexes have a less dense core compared to polyplexes based on cationic homopolymers (such as PEI) as reported before. The polyplexes showed to have a dynamic character meaning that the polymer chains can exchange between the polyplex core and shell. Mobility of the polymers allow their uniform redistribution within the polyplex and this feature has been reported to be favorable in the context of pDNA release and subsequent improved transfection efficiency, compared to nondynamic formulations

Sustained Release of Vascular Endothelial Growth Factor from Poly(ε-caprolactone-PEG-ε-caprolactone)‑b‑Poly(L‑lactide) Multiblock Copolymer Microspheres

Vascular endothelial growth factor (VEGF) is the major regulating factor for the formation of new blood vessels, also known as angiogenesis. VEGF is often incorporated in synthetic scaffolds to promote vascularization and to enhance the survival of cells that have been seeded in these devices. Such applications require sustained local delivery of VEGF of around 4 weeks for stable blood vessel formation. Most delivery systems for VEGF only provide short-term release for a couple of days, followed by a release phase with very low VEGF release. We now have developed VEGF-loaded polymeric microspheres that provide sustained release of bioactive VEGF for 4 weeks. Blends of two swellable poly(ε-caprolactone)−poly(ethylene glycol)−poly(ε-caprolactone)-b-poly(L-lactide) ([PCL−PEG−PCL]-b-[PLLA])-based multiblock copolymers with different PEG content and PEG molecular weight were used to prepare the microspheres. Loading of the microspheres was established by a solvent evaporation-based membrane emulsification method. The resulting VEGF-loaded microspheres had average sizes of 40−50 μm and a narrow size distribution. Optimized formulations of a 50:50 blend of the two multiblock copolymers had an average VEGF loading of 0.79 ± 0.09%, representing a high average VEGF loading efficiency of 78 ± 16%. These microspheres released VEGF continuously over 4 weeks in phosphate-buffered saline pH 7.4 at 37 °C. This release profile was preserved after repeated and long-term storage at −20 °C for up to 9 months, thereby demonstrating excellent storage stability. VEGF release was governed by diffusion through the water-filled polymer matrix, depending on PEG molecular weight and PEG content of the polymers. The bioactivity of the released VEGF was retained within the experimental error in the 4-week release window, as demonstrated using a human umbilical vein endothelial cells proliferation assay. Thus, the microspheres prepared in this study are suitable for embedment in polymeric scaffolds with the aim of promoting their functional vascularization