Release Of Fluorescein Research Articles

Mucoadhesive poly(ethylene glycol)-based biodegradable polyesters with in-chain model drug

Mucoadhesive polymers have received significant interest in drug delivery due to the prolonged residence time on various mucus gel layers. Not only local effects but also the systemic uptake of active pharmaceutical ingredients can be enhanced by such mucoadhesive excipients. In this study, we design novel poly(ethylene glycol) based mucoadhesive polyesters bearing unsaturated or sulfhydryl functionalities and model drug fluorescein polymerized in the polymeric backbone. The structure of polyesters was confirmed via 1H NMR and FTIR, while their molar masses, determined by gel permeation chromatography, were found to be between 3.6 and 4.0 kDa. Enzymatic degradation studies showed the ability of lipase to degrade the polymeric chains. Because of the mucoadhesive groups, the viscosity of mucus mixtures with these polymers increased 1.8-fold for the double bond, while 4.3-fold for thiol functional polymers, compared to PEG. Prolonged residence time on porcine intestinal mucosa was detected, as 3 h after application, up to 69 % of the polymers were still on the mucosa, whereas PEG was completely removed within 30 mins. Highly pH and enzyme-dependent release of fluorescein was noticed; only pH > 6.8 caused significant drug release. According to the results of this study, PEG-based unsaturated and thiolated polyesters with in-chain model drugs are great mucoadhesive and biodegradable drug carriers with sustained release properties.

Kinetic Model of Fluorescein Release through Bioprinted Polylactic Acid Membrane.

Polylactic acid (PLA)-based cylindrical membranes for the controlled release of fluorescein sodium salt (FS) were prepared by bioprinting on systems with an initial FS concentration of 0.003763 gdm-3 and 37.63 gdm-3, and the drug release process was monitored in a bath at 37 °C. Photographs, acquired at regular intervals during the process, revealed marked osmotic swelling of the polymer. Osmotic swelling consists in the enlargement of the polymer structure and due to the influx of water molecules across the membrane. The cylindrical PLA membrane starts to significantly swell once a certain threshold range is crossed. Important amounts of FS can dissolve under these radically changed circumstances, and the dissolved FS molecules are mobile enough to diffuse out of the cylinder, thus allowing drug release. As a matter of fact, in this investigation, we ascertained that polymer swelling promotes the mass transport phenomenon by altering the conditions for drug dissolution and diffusion, hence facilitating FS release after a specific lag time. Furthermore, in order to compare the release kinetics, the half-release time, t0.5, was taken into consideration. The data of this study evidence that, while increasing the initial concentration of FS by three orders of magnitude, the time parameter, t0.5, is only reduced by 5/6. In addition, the yield of the release process is drastically reduced due to the strong aggregation ability of the dye. Finally, it is demonstrated that a compressed exponential kinetic model fits the experimental data well despite the varying physical conditions.

New non‐toxic fluorescent marker for quantifying the viability of corneal endothelial cells

Aims/Purpose: Viability markers are essential for cellular research and tissue transplantation. However, existing options such as Trypan Blue lack specificity, and some clinically used markers are cytotoxic. Therefore, we synthesized the compound F4267, a non‐fluorescent marker that releases fluorescein upon hydrolysis. The aim of this study was to evaluate the quality of F4267 labelling of endothelial cells and its cytotoxicity in immortalized cells and organ‐cultured endothelium.Methods: We compared the marking capability of F4267 and Calcein‐AM using immortalized cells and endothelium from corneal pairs. The quality of fluorescent labelling was assessed using F4267 at 4 μM and Calcein‐AM at 4 μM. Cytotoxicity of F4267 was evaluated at varying concentrations of 40 and 1000 μM on cells, and at 40 μM on corneal endothelium, with single, repeated, or prolonged exposures. The final density and viability of corneal endothelium were measured using HEC1 staining.Results: F4267 (40 μM) exhibited fluorescent labelling quality equivalent to Calcein‐AM (4 μM), allowing clear differentiation between living, dying, and acellular areas. In HCE‐2 cells, cytotoxicity was reduced by 19% with F4267 (40 μM) compared to Calcein‐AM (4 μM). Prolonged exposure (24 h) to HCEC‐B4G12 cells showed no cytotoxicity with F4267 (40 and 100 μM), but resulted in destruction of the cell layer with Calcein‐AM (4 μM). F4267 (40 μM) also demonstrated significantly lower cytotoxicity, reducing mortality by 10% (single exposure) and 17% (repeated exposure) in organ‐cultured corneas.Conclusions: F4267 shows promise as a marker for assessing viable endothelial cells in organ culture, providing a safe alternative to existing markers. Its release of fluorescein with excellent cell tolerance supports its clinical potential. The integration of viable endothelial cell density assessment would enhance the value of corneal banks. Further validation is required, as well as exploration of broader applications.Reference1. Pipparelli et al., IOVS 2011.

Gated nanoprobe utilizing metal-organic frameworks for identifying and distinguishing between the wild strains and the vaccine strains of brucella.

In the assay for Brucella, the identification and differentiation of wild strains and vaccine strains present a significant challenge. Currently, there aren't any commercially available product to address this issue. In this study, we have developed a novel gated nanoprobe by utilizing Metal-Organic Frameworks (MOFs) as a scaffold and hairpin DNA as a "gating switch". Specifically, Probe 1 with hairpin structure (P1h) targets a gene that is present in both wild strains Y3 (B. melitensis biovar 3) and vaccine strains A19 (Brucella abortus strains A19). We successfully applied this probe to screen positive samples of Brucella without any cross-reactivity with other substances. Additionally, we identified another specific gene exclusively found in wild strains, which serves as Probe 2 with hairpin structure (P2h) to confirm the strain type. Simultaneous detachment of both P1h and P2h from the MOFs leads to the release of Rhodamine 6G (Rho 6G) and Fluorescein (Flu), specifically indicating the presence of wild strains. If only P1h detaches and the Flu signal is detected, it suggests the presence of vaccine strains. Importantly, this method offers high accuracy, with a detection rate of 90% and a recovery rate of 94.71% to 107.65%, while avoiding cross-reactions with MO and TB. This one-step experiment provides reliable identification and differentiation of Y3 and A19, addressing concerns related to long periodicity, interference from individual variations, and the complex design of primers in existing laboratory methods. Furthermore, our approach successfully detects target 1 (T1) and target 2 (T2) at concentrations ranging from 10-6 M to 10-9 M, with a detection limit of 6.7 × 10-10 M and 6.4 × 10-10 M, respectively. Importantly, our strategy is cost-effective (around $1) and offers higher detection efficiency compared to traditional laboratory methods.

Decasubstituted Pillar[5]arene Derivatives Containing L-Tryptophan and L-Phenylalanine Residues: Non-Covalent Binding and Release of Fluorescein from Nanoparticles.

Sensitive systems with controlled release of drugs or diagnostic markers are attractive for solving the problems of biomedicine and antitumor therapy. In this study, new decasubstituted pillar[5]arene derivatives containing L-Tryptophan and L-Phenylalanine residues have been synthesized as pH-responsive drug nanocarriers. Fluorescein dye (Fluo) was loaded into the pillar[5]arene associates and used as a spectroscopic probe to evaluate the release in buffered solutions with pH 4.5, 7.4, and 9.2. The nature of the substituents in the pillar[5]arene structure has a huge influence on the rate of delivering. When the dye was loaded into the associates based on pillar[5]arene derivatives containing L-Tryptophan, the Fluo release occurs in the neutral (pH = 7.4) and alkaline (pH = 9.2) buffered solutions. When the dye was loaded into the associates based on pillar[5]arene with L-Phenylalanine fragments, the absence of release was observed in every pH evaluated. This happens as the result of different packing of the dye in the structure of the associate. This fact was confirmed by different fluorescence mechanisms (aggregation-caused quenching and aggregation-induced emission) and association constants. It was shown that the macrocycle with L-Phenylalanine fragments binds the dye more efficiently (lgKa = 3.92). The experimental results indicate that the pillar[5]arene derivatives with amino acids fragments have a high potential to be used as a pH-responsive drug delivery devices, especially for promoting the intracellular delivering, due to its nanometric size.

Development of Fluorescently Labeled Self-Emulsifying Drug Delivery Systems (SEDDS) for Prolonged Stability, In Vitro Sustained Release, and Cellular Uptake.

In this study, four fluorescein hydrophobic ionic complexes were formed with the cationic polymers Eudragit RS, Eudragit RL, Eudragit E, and polyethyleneimine (PEI) to provide fluorescein sustained release, sustained cellular uptake, and stability. Complexes were loaded in a self-emulsifying drug delivery system (SEDDS) composed of 40% Tween 80, 20% Kolliphor EL, 15% 2-n-Octyl-1-dodecanol, and 25% dipropylene glycol. SEDDS were investigated regarding their size, polydispersity index (PDI), zeta potential, and cytotoxicity. Fluorescein release from SEDDS was performed in phosphate buffer (pH 6.8 and pH 8), and the released fluorescein was evaluated for cellular uptake. Moreover, fluorescein from all of the SEDDS pre-concentrates was released at different time points to check its long-term stability over six months. The average fluorescein load in SEDDS was 0.045%. SEDDS showed an average droplet size of 24.9 ± 1.6 nm with PDI ≤ 0.3. SEDDS complexes diluted 1:100 increased the zeta potential from -7.3 mV to +3.7 mV and provided > 85% cell viability. A 92.27 ± 3.18% fluorescein exhibited a few seconds of immediate release when used as control or PEI complex in SEDDS. On the contrary, Eudragit-fluorescein complexes in SEDDS showed sustained release of 87.01 ± 5.22% fluorescein in ≤ 70 min with 22.19 ± 14.56% and 59.27 ± 16.57% released at 10 min in pH 6.8 and pH 8 release media, respectively. Comparatively, the medium at pH 6.8 maintained a significantly improved sustained fluorescein release (p ≤ 0.001). Furthermore, Eudragit RS/RL compared to Eudragit E, significantly exhibited a slower fluorescein release rate from SEDDS (p ≤ 0.01). The cellular uptake of the released fluorescein was 72.4 ± 8.2% for all SEDDS complexes after 3 h. Eudragit complexes compared to PEI complex in SEDDS significantly showed m ore sustained fluorescein cellular uptake at 1 h and 2 h (p ≤ 0.001). However, SEDDS complexes showed the longest fluorescein stability with PEI after six months, whereas fluorescein stability for SEDDS containing fluorescein as Eudragit complex and control showed 39.1% and 82.5% fluorescence decrease, respectively, after three months. In the developed SEDDS, the presence of hydrophobic ionic complexes can significantly promote longer stability and sustained cellular uptake of fluorescein while releasing in a sustained manner.

Molecular Study of Ultrasound-Triggered Release of Fluorescein from Liposomes.

Several investigations have suggested that ultrasound triggers the release of drugs encapsulated into liposomes at acoustic pressures low enough to avoid cavitation or high hyperthermia. However, the mechanism leading to this triggered release as well as the adequate composition of the liposome membrane remains unknown. Here, we investigate the ultrasound-triggered release of fluorescein disodium salt encapsulated into liposomes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-distearoylphosphatidyl-ethanolamine (DSPC) lipids with various concentrations of cholesterol (from 0 to 44 mol %). The passive release of encapsulated fluorescein was first characterized. It was observed to be higher when the membrane is in a fluid phase and increased with temperature but decreased upon addition of cholesterol. Next, the release of fluorescein was measured at different acoustic frequencies (0.8, 1.1, and 3.3 MHz) and peak-to-peak pressures (0, 2, 2.5, 5, and 8 MPa). Measurements were performed at temperatures where DOPC and DSPC liposomes were, respectively, in the fluid or gel phase. We found that the release rate did not depend on the ultrasound frequency. For DOPC liposomes, the ultrasound-triggered release of fluorescein decreased with increasing concentration of cholesterol in liposomes, while the behavior was more complex for DSPC liposomes. Overall, the triggered release from DSPC liposomes was up to ten times less than DOPC liposomes. Molecular dynamics simulations performed on a pure DOPC membrane showed that a membrane experiences, under a directional pressure of ±2.4 MPa, various changes in properties such as the area per lipid (APL). An increase in the APL was notably observed when the simulation box was laterally stretched or perpendicularly compressed, which was accompanied by an increase in the number of water molecules crossing the membrane. This suggests that ultrasound most probably enhances the diffusion of encapsulated molecules at small acoustic pressures by increasing the distance between lipids.

Hydroxyapatite sonosensitization of ultrasound-triggered, thermally responsive hydrogels: An on-demand delivery system for bone repair applications.

While bones have the innate capability to physiologically regenerate, in certain cases regeneration is suboptimal, too slow, or does not occur. Biomaterials-based growth factor delivery systems have shown potential for the treatment of challenging bone defects, however, achieving controlled growth factor release remains a challenge. The objective of this study was to develop a thermally responsive hydrogel for bone regeneration capable of ultrasound-triggered on-demand delivery of therapeutic agents. Furthermore, it was hypothesized that incorporation of hydroxyapatite (HA) into the hydrogel could increase sonosensitization, augmenting ultrasound sensitivity to enable controlled therapeutic release to the target tissue. Alginate thermally responsive P(Alg-g-NIPAAm) hydrogels were fabricated and varying quantities of HA (1, 3, 5, and 7% wt./vol.) incorporated. All hydrogels were highly injectable (maximum injection force below 6.5N) and rheological characterization demonstrated their ability to gel at body temperature. The study demonstrated the ultrasound-triggered release of sodium fluorescein (NaF), bovine serum albumin (BSA), and bone morphogenetic protein 2 (BMP-2) from the hydrogels. Release rates of BSA and BMP-2 were significantly enhanced in the HA containing hydrogels, confirming for the first time the role of HA as a son sensitizer. Together these results demonstrate the potential of these ultrasound-triggered thermally responsive hydrogels for on-demand delivery of therapeutic agents for bone regeneration.

Potato Starch Edible Films as Environmentally Friendly Carriers for Model Drug: In vitro Release Study

Starch is a vital plant polymer used in thousands of food and non-food products. Researchers have made great efforts over the past 20 years to develop ingredients based on nature which enhance starch texture and nutritional values. Starch has many non-food applications, ranging from body care to medicinal applications, among its uses in other foodstuffs. Since starch is sustainable and environmentally friendly material, in many chemical applications, including plastics, detergents and glues, it can serve as a good substitute for fossil-fuel components. This work aims to introduce edible starch films-based carriers for supporting the release of fluorescein as a model drug. Physical modification for potato starch carriers (PSS) was done by incorporating glycerol and low-density polyethylene (LDPE) molecules. Fluorescein dye was incorporated in edible films to investigate the amount of release by UV-vis technique. It was found that PSS-LDPE is spread better than LDPE at physiological temperature and PSS dissolution is also appropriate at this temperature. While PSS formed faster edible film than LDPE and PSS-LDPE, it decayed within 3 days. The amounts of dye released from PSS and PSS-LDPE films were higher than that of fluorescein dispersed in starch suspension in buffer solution (pH 7.5) at 37 o C.

Preparation of dimethylaminopropyl octadecanamide/stearic acid vesicles incorporating azobenzene and their UV-responsive release property

UV-responsive vesicle was prepared by incorporating azobenzene in the vesicular membrane composed of dimethylaminopropyl octadecanamide (DMAPODA) and stearic acid (SA). Azobenzene intercalated in the vesicular membrane underwent cis-to-trans isomerization under the UV irradiation, confirmed by UV spectroscopy. The phase transition temperature of vesicle was found to be around 51.2 °C by differential scanning calorimetry. Using a negative staining technique, the vesicles were found as circular and irregular-shaped particles on the TEM photo. The mean hydrodynamic diameter of vesicles increased from 199.4 to 1597.3 nm when the lipid/azobenzene ratio increased from 100/0 to 100/5. The zeta potential of DMAPODA/SA vesicle decreased from about + 60 mV to – 60 mV when the pH value of medium increased from 3.0 to 10.0, and the inclusion of azobenzene in the vesicular membrane had little effect on the zeta potential. UV irradiation markedly promoted the release of carboxylic fluorescein loaded in vesicle incorporating azobenzene, possibly because the cis-to-trans photoisomerization would disturb the well-ordered crystalline structure of the vesicular membrane.

Triggered Small-Molecule Release from Dual-Stimuli Responsive Microgels

Light- and temperature-responsive poly(N-isopropylacrylamide-co-nitrobenzyl methacrylate) (pNIPAm-co-NBMA) microgels were synthesized via free-radical precipitation polymerization and exhibited a hydrodynamic diameter of 320 ± 40 nm. We demonstrated that the microgels’ NMBA groups were cleaved upon UV irradiation, which we showed could be used for light-activated release of fluorescein (used as a model drug molecule). Moreover, drug release could also be promoted by microgel deswelling due to heating. Specifically, the hydrophobic model drug fluorescein was incorporated into the pNIPAm-co-NBMA microgels via physical interactions, and we showed that exposure to UV light (or heating) could induce its release. Thus, the pNIPAm-co-NBMA microgels could find the use for applications requiring triggered release of cargo when needed.

Soft Template Electropolymerization of Polypyrrole for Improved pH-Induced Drug Delivery.

Chronic wounds are characterized by a localized pH change from acidic (healthy) to alkaline (unhealthy), which can be harnessed to act as a switch for drug release from a polymer medium covering the wound for improved healing. To realize this, a new polymer dressing material is needed to help heal chronic wounds. Polypyrrole (PPy) is a biocompatible electroactive polymer that has been proven as a successful drug delivery mechanism, but currently lacks the capacity for scalable clinical applications due to its poor processability. In this study, PPy films with and without microstructures were produced using electrochemical oxidation and subsequently doped with fluorescein, a model drug molecule. To increase the drug loading capacity, microstructures were created through soft template polymerization of pyrrole around hydrogen gas bubbles. Fluorescein release was measured using UV spectroscopy over a pH range of 2 to 11, showing increased release at higher pH values. Microstructured films showed an increased doping capacity compared to flat PPy films, attributed to the increase in drug incorporation sites. The pH-activated release mechanism was shown to be successful and can be applied as a pH-sensitive biosensor and drug delivery system in vitro.

Kinetic analysis of pH influence on the fluorescein release from chitosan-coated mesoporous silica nanoparticles

The development of drug delivery systems that are able to respond to the different pH gradients found in the human body is a research field of vast scientific and technological interests. With such a responsive system, a specific pH condition can be used as a stimulus to trigger the local drug-releasing, increasing the efficiency of treatments in comparison to the traditional ones of systemic drug administration. In this study, a responsive drug nanocarrier to acid pH was produced by coating mesoporous silica nanoparticles with an outer chitosan layer that was covalently bonded to the nanoparticle. Fluorescein dye (FL) was loaded into the nanocarrier and used as a spectroscopic probe to evaluate the kinetics of release in buffered solutions with pH 6.0, 7.2 and 8.0. The kinetics analysis using mathematical models demonstrated higher rates of release in pH 6.0 than in pH 7.2 or 8.0. However, the experimental procedure used to load the dye into the nanocarrier had a huge influence on the kinetic mechanisms of delivering. When the dye was loaded into the nanocarrier before the chitosan coating had been made, the FL release was sustained and controlled through a Fickian's transport mechanism. When the dye was loaded after the nanocarrier coating a burst release effect was observed in every pH evaluated, which indicated that the dye was loaded only in the external coating of CS and did not inside of the silica mesopores.

Electrochemically Controlled Drug Release from a Conducting Polymer Hydrogel (PDMAAp/PEDOT) for Local Therapy and Bioelectronics.

In this study, the release of fluorescein from a photo-crosslinked conducting polymer hydrogel made from a hydrogel precursor poly(dimethylacrylamide-co-4-methacryloyloxy benzophenone (5%)-co-4-styrenesulfonate (2.5%)) (PDMAAp) and the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) is investigated. Fluorescein, here used as a model for a drug, is actively released through application of an electrical trigger signal. The detected quantity is more than six times higher in comparison to that released from a conventional PEDOT/polysterene sulfonate (PSS) system. Release profiles, drug dose, and timing can be tailored by the application of different trigger signals and pretreatments. To demonstrate that the novel drug release system can be used for a drug relevant for local delivery to a neural interface, experiments are furthermore performed with the anti-inflammatory drug dexamethasone (Dex). The conducting polymer hydrogel facilitates the active release of Dex, in comparison to the previously used PEDOT/Dex. It is suggested that PEDOT/PDMAAp is an interesting alternative for conducting polymer based drug release systems, with the potential to offer more volume for storage, yet retaining the excellent electrochemical properties known for PEDOT electrodes.

Standing acoustic waves in microfluidic channels for enhanced intracellular delivery of molecular compounds

Intracellular delivery of molecular compounds is required for many in vitro research applications. Ultrasound-induced cavitation has been shown to enhance intracellular delivery of molecular compounds via mechanisms that may include sonoporation or endocytosis. Recently, acoustofluidic approaches have been developed to utilize standing acoustic waves (SAW) for cell manipulation in microfluidic channels. In this study, the effect of SAW on fluorescein release from perfluorocarbon double emulsion droplets in microfluidic channels was explored. Vaporization of perfluorocarbon double emulsion droplets induced by SAW may potentially enhance intracellular delivery of molecular compounds. In this study, fluorescein-loaded double emulsions were passed through a microfluidic device and exposed to 8-MHz SAW. Fluorescein release was quantified by measuring the change in fluorescence of the supernatant before and after treatment. Treatment with SAW in microfluidic channels increased the fluorescence by 8.8-fold compared to the baseline level. Fluorescein release was also higher after treatment with SAW compared to samples that passed through the microfluidic device without exposure to SAW (p = 0.03). These results suggest that SAW and perfluorocarbon double emulsions in microfluidic channels could potentially enhance the efficiency and consistency of intracellular molecular delivery in vitro.

Microbial decomposition of porcine tissue in organic compost at different temperatures

Laboratory experiments were conducted to determine the effect of temperature (20 °C and 30 °C) on the rate of tissue decomposition in organic compost for a period of 28 days. Porcine tissue was used as a substitute for human body parts in burial microenvironment. Measurements of the decay include porcine tissue mass loss, soil pH, the metabolic activity of soil microbes, viable count and Gram stain of associated microflora. Incubation temperature had a significant impact on the post-mortem decay rates and stages at 30 °C in comparison to the samples incubated at 20 °C. Bacterial enumeration demonstrated microbial burden to be higher at higher temperatures, suggesting accelerated decomposition. Gram staining identified mostly Gram-negative bacteria, decomposers that might have originated from the organic meat as opposed to soil bacteria. There was a significant difference in soil pH levels during harvest times and at the end of the experiments in both microenvironments. It is an indication that measured pH of the depository may reveal whether the body parts were recently buried. A fluorescein diacetate (FDA) method that measured metabolic activity in compost demonstrated the highest release of fluorescein during harvests at 30 °C. A non-parametric Friedman test of differences among repeated measures rendered significant results (α=0.00) showing differences in microbial activity according to the temperature level. The results suggest that depositional microenvironment would be significantly modified by the decaying organic matter with the rise of temperature. This way, the temperature would make an impact on the decay of buried remains.

Mathematical Models as Tools to Predict the Release Kinetic of Fluorescein from Lyotropic Colloidal Liquid Crystals.

In this study, we investigated the release kinetic of fluorescein from colloidal liquid crystals made from monoglyceride and different non-ionic surfactants. The crystals were physicochemically characterized and the release experiments were carried out under the sink conditions, while mathematical models were described as extrapolations from solutions of the diffusion equation, in different initial and boundary conditions imposed by pharmaceutical formulations. The diffusion equation was solved using Laplace and Fourier transformed functions for release kinetics from infinite reservoirs in a semi-infinite medium. Solutions represents a general square root law and can be applied for the release kinetic of fluorescein from lyotropic colloidal liquid crystals. Akaike, Schwartz, and Imbimbo criteria were used to establish the appropriate mathematical model and the hierarchy of the performances of different models applied to the release experiments. The Fisher statistic test was applied to obtain the significance of differences among mathematical models. Differences of mathematical criteria demonstrated that small or no significant statistic differences were carried out between the various applied models and colloidal formulations. Phenomenological models were preferred over the empirical and semi-empirical ones. The general square root model shows that the diffusion-controlled release of fluorescein is the mathematical models extrapolated for lyotropic colloidal liquid crystals.

Fluorogenic approach to evaluating prodrug hydrolysis and stability in live cells.

Fluorescein diester which is conjugated with cell membrane permeable Arg9 peptide was proposed as probe for ester prodrug stability and drug release study in living cells. α-Amino protected d-Val and l-Ala which bear differently hindered side chains were used to afford model diesters of 5-maleimide-fluorescein. Such fluorescein diesters were further conjugated with a Cys containing cell membrane permeable Arg9 peptide via thiol-ene crosslink reaction. The resulted conjugates of fluorescein diester and Arg9 peptide were purified with HPLC and characterized with MALDI-TOF MS. Upon incubation with cultured cells, the fluorescein diesters were delivered into the cells, the following hydrolysis of fluorescein diesters and release of fluorescein inside living cells were observed by monitoring the fluorescence accumulation. Fluorescence microscopic imaging studies of HeLa cells treated with fluorescein l-Ala diester show strong fluorescence accumulation in 30 min indicating fast hydrolysis of fluorescein diester and fluorescein release; in contrast d-Val diester remains stable inside cells evidenced by margin fluorescence formation. Further flowcytometry studies on the fluorescein diester-Arg9 conjugate treated cells show that the hydrolysis t1/2 for l-Ala diester is 15 min. The results also show that Arg9 peptide not only transports the ester probes into cell efficiently but also can retain and concentrate hydrolytic product fluorescein inside cells so that the accumulated fluorescence can be accurately quantified. This fluorogenic probe approach provides feasible applications in dynamic studies on ester prodrug hydrolysis and release, facilitating screening and optimization of prodrug structures in living cell settings.

Magnetic Heating Stimulated Cargo Release with Dose Control using Multifunctional MR and Thermosensitive Liposome.

Rationale: Magnetic resonance imaging (MRI) is one of the most widely used diagnostic tools in the clinic. In this setting, real-time monitoring of therapy and tumor site would give the clinicians a handle to observe therapeutic response and to quantify drug amount to optimize the treatment. In this work, we developed a liposome-based cargo (cancer drugs) delivery strategy that could simultaneously monitor the real-time alternating magnetic field-induced cargo release from the change in MRI relaxation parameter R1 and the location and condition of liposome from the change in R2. The tumor site can then be monitored during the cargo release because liposomes would passively target the tumor site through the enhanced permeability and retention (EPR) effect. Physical insights from the experimental results and corresponding Monte Carlo spin dynamics simulations were also discussed.Methods: Superparamagnetic iron oxide (SPIO) nanoparticles, diethylenetriaminepentaacetic acid gadolinium(III) (Gd(III)-DTPA), and a model cancer drug (fluorescein) were co-loaded in PEGylated thermosensitive liposomes. The liposomes were characterized by transmission electron cryo-microscopy (cryoTEM), dynamic light scattering (DLS), and inductively coupled plasma optical emission spectrometry (ICP-OES). Alternating magnetic field (AMF) was used to create controlled mild hyperthermia (39-42°C) and facilitate controlled cargo (fluorescein) release from the thermosensitive liposomes. MRI relaxation parameters, R1 and R2, were measured at room temperature. The temporal variation in R1 was used to obtain the temporal profile of cargo release. Due to their similar sizes, both the gadolinium and cargo (model cancer drug fluorescein) would come out of the liposomes together as a result of heating. The temporal variation in R2 was used to monitor SPIO nanoparticles to enhance the tumor contrast. Monte Carlo spin dynamics simulations were performed by solving the Bloch equations and modeling SPIO nanoparticles as magnetized impenetrable spheres.Results: TEM images and DLS measurements showed the diameter of the liposome nanoparticle ~ 200 nm. AMF heating showed effective release of the model drug. It was found that R1 increased linearly by about 70% and then saturated as the cargo release process was completed, while R2 remained approximately constant with an initial 7%-drop and then recovered. The linear increase in R1 is consistent with the expected linear cargo release with time upon AMF heating. Monte Carlo spin dynamics simulations suggest that the initial temporal fluctuation of R2 is due to the plausible changes of SPIO aggregation and the slow non-recoverable degradation of liposomal membrane that increases water permeability with time by the heating process. The simulations show an order of magnitude increase in R2 at higher water permeability.Conclusion: We have performed MR parameter study of the release of a cargo (model cancer drug, fluorescein) by magnetic heating from thermosensitive multifunctional liposomes loaded with dual contrast agents. The size of the liposome nanoparticles loaded with model cancer drug (fluorescein), gadolinium chelate, and SPIO nanoparticles was appropriate for a variety of cancer therapies. A careful and detailed analysis with theoretical explanation and simulation was carried out to investigate the correlation between MRI relaxation parameters, R1 and R2, and different cargo release fractions. We have quantified the cargo release using R1, which shows a linear relation between each other. This result provides a strong basis for the dosage control of drug delivered. On the other hand, the fairly stable R2 with almost constant value suggests that it could be used to monitor the position and condition of the liposomal site, as SPIO nanoparticles mostly remained in the aqueous core of the liposome. Because our synthesized SPIO-encapsulated liposomes could be targeted to tumor site passively by the EPR effect, or actively through magnetofection, this study provides a solid ground for developing MR cancer theranostics in combination of this nanostructure and AMF heating strategy. Furthermore, our simulation results predict a sharp increase in R2 during the AMF heating, which opens up the exciting possibility of high-resolution, high-contrast real-time imaging of the liposomal site during the drug release process, provided AMF heating could be incorporated into an MRI setup. Our use of the clinically approved materials, along with confirmation by theoretical simulations, make this technique a promising candidate for translational MR cancer theranostics.

Click and photo-release dual-functional nucleic acid nanostructures.

We functionalize nucleic acid nanostructures with click chemistry (for attachment of cargos) and a photocleavable linker (for release). We demonstrate cargo attachment using a fluorescein dye and release using UV trigger from an RNA three-way junction, a DNA star motif and a DNA tetrahedron. Such multifunctional nucleic acid nanostructures have potential in targeted drug delivery.