A stimulus-responsive, in situ-forming, nanoparticle-laden hydrogel for ocular drug delivery
Most medications targeting optic neuropathies are administered as eye drops. However, their corneal penetration efficiencies are typically < 5%. There is a clear, unmet need for novel transcorneal drug delivery vehicles. To this end, we have developed a stimulus-responsive, in situ-forming, nanoparticle-laden hydrogel for controlled release of poorly bioavailable drugs into the aqueous humor of the eye. The hydrogel is formulated as a composite of hyaluronic acid (HA) and methylcellulose (MC). The amphiphilic nanoparticles are composed of poly(ethylene oxide) (PEO) and poly(lactic acid) (PLA). Experimental design aided the identification of hydrogel composition and nanoparticle content in the formulation, and the formulation reliably switched between thixotropy and temperature-dependent rheopexy when it was tested in a rheometer under conditions that simulate the ocular surface, including blinking. These properties should ensure that the formulation coats the cornea through blinking of the eyelid and facilitate application of the medication as an eye drop immediately prior to the patient’s bedtime. We subsequently tested the efficacy of our formulation in whole-eye experiments by loading the nanoparticles with cannabigerolic acid (CBGA). Our formulation exhibits over a 300% increase in transcorneal penetration over control formulations. This work paves the way for the introduction of novel products targeting ocular diseases to the market.
KeywordsCannabinoids Glaucoma Hydrogel Nanoparticles Synthetic biology Switchable rheology
The space enclosed by the cornea and iris is referred to as the anterior chamber of the eye. The base of the cornea extends into a tissue called the ciliary body, and this interface is aligned by a spongy tissue called the trabecular meshwork . Epithelial cells in the ciliary body secrete a thin, gelatinous fluid known as the aqueous humor that permeates through the trabecular meshwork to fill the anterior chamber. As freshly secreted aqueous humor makes its way into the chamber, older fluid drains out, also through the meshwork. The constant circulation of aqueous humor through the trabecular meshwork is central to eye hygiene and health. Likewise, the posterior chamber of the eye, which is enclosed by the lens and the retina, is filled with a viscous, jelly-like fluid called the vitreous humor. Inadequate or obstructed drainage of the aqueous humor through the trabecular meshwork, which is the pathophysiology of glaucoma, increases the fluid pressure within the anterior chamber, which subsequently propagates to the posterior chamber of the eye . The increased intraocular pressure exacts a toll on the basal membrane of the retina, thinning the mesh-like tissue in this region and damaging the head of the optic nerve housed therein by inducing apoptotic degeneration of the nerve’s ganglion cells.
Chronic optic neuropathies such as glaucoma are the leading causes of blindness worldwide . It is estimated that nearly 80 million people will be affected by these conditions by 2020 . Most neuroprotective medications for treating optic neuropathies are administered as eye drops. However, since less than 5% of the drug in eye drops actually penetrates the cornea [5, 6], the dosage of drug in the formulation is typically much higher than what is required, which implies that eye drops are fairly expensive to manufacture. Additionally, once the neuroprotective drug penetrates the cornea and lens, it must then diffuse through the aqueous and vitreous humors before it can interact with receptors on the surface of retinal ganglion cells. These barriers to mass transport further lower the efficacy of drugs administered as eye drops. There is considerable room for improvement for drug formulations for ophthalmological applications, in particular, new and effective delivery vehicles for neuroprotective drugs.
Our therapeutic strategy leverages the proven potential of cannabinoids to confer neuroprotection to ganglion cells . Although the role of cannabinoids in treating glaucoma is well understood, no such products currently exist in the market. Current glaucoma remedies work by lowering intraocular pressure either by inhibiting carbonic anhydrase in the eye, or reducing the production of aqueous humor by the ciliary epithelial cells, or by increasing fluid drainage through the trabecular meshwork . Likewise, devices that provide a conduit for the intraocular fluid to drain into the nasolacrimal system have also been tested for the treatment of glaucoma . The nasolacrimal drainage system discharges tear fluid from the external eye into the nasal cavity and plays key roles in maintenance of eye hygiene and clearance of drugs. Neuroprotection as a therapeutic strategy has not hitherto been pursued owing to great difficulties associated with the targeted delivery of cannabinoids to the base of retina and the poor bioavailability of these molecules . Previous attempts for topical delivery of cannabinoids to human ocular tissues were limited to using mineral oil  or cyclodextrins [12, 13, 14, 15]. However, these formulations are either cytotoxic or irritate the ocular tissue . The development of a superior drug delivery vehicle would surely pave the way to market for non-invasive, cannabinoid-based neuropathic pain relievers for treating glaucoma and other chronic optic neuropathies.
To this end, we have developed a stimulus-responsive, in situ-forming, nanoparticle-laden hydrogel for spatiotemporal and dosage-controlled release of poorly bioavailable cannabinoids into the aqueous humor of the eye. The hydrogel is formulated as a composite of hyaluronic acid (HA) and methylcellulose (MC). The former is an anionic polysaccharide, and the latter is a water-soluble, hydrophobic derivative of cellulose. Both materials are biocompatible and highly mucoadhesive and have been recognized as safe by the FDA [17, 18]. In order to enhance the flux of the drug through the cornea and sustain its release for a prolonged duration, we also employed nanoparticles as the drug carriers. The nanoparticles (NPs) are composed of poly(ethylene oxide) (PEO) and poly(lactic acid) (PLA) and are loaded with cannabigerolic acid (CBGA), a molecule that is a close mimic of the cannabinoids that have been approved for use in glaucoma medication. Additionally, we synthesized the CBGA by expressing the cannabinoid biosynthetic pathway in Escherichia coli, and the drug-loaded NPs were synthesized using nanoprecipitation. Since we desired rapid gelation of the formulation upon contact with the ocular surface and formation of a uniform, unintrusive coating over the cornea that can be positioned by blinking of the eyelid, we implemented a factorial design of experiments in order to identify the constitution of the formulation that allows it to switch between temperature-dependent rheopexy (thickening) and thixotropy (thinning). Parameters that were investigated include the concentration of HA and MC, the size of the NPs, and the drug loading, among others. The optimized formulation has a sol-gel transition temperature of roughly 32 °C, which is comparable with that of the ocular surface, and successfully transitioned between shear thinning and temperature thickening when it was tested in a rheometer under conditions that simulated the ocular surface. Finally, we tested the efficacy of our formulation in whole-eye experiments using porcine eyeballs. Our work seamlessly combines product design, synthetic biology, polymer rheology and analysis of mass transport within ocular tissue, and the formulation that we have developed exhibits over a 300% increase in transcorneal penetration over control formulations. Moreover, the nanoparticle-laden hydrogel can be packaged as a liquid, which permits easy dosing and manufacturability. The current study paves the way to the market for cannabinoid-based drugs, as well as combination therapies with existing intraocular pressure-lowering pharmaceuticals.
Sodium hyaluronate (MW 752 kDa) was purchased from Lifecore Biomedical LLC (Chicago, IL). Methylcellulose (MC A15 PREM LV) was obtained as a gift from Dow Chemical (Midland, MI). PEO-b-PLA (5.0-b-23.0) was purchased from Polymer Source Inc. (Montreal, QC). Carboxy-functionalized poly(styrene) NPs bearing a range of sizes were purchased from Phosphorex (Hopkinton, MA). Olivetolic acid (OA) was synthesized as described previously , and all reagents were purchased from Sigma Aldrich Canada (Oakville, ON). Geranyl pyrophosphate (GPP) was also purchased from Sigma Aldrich Canada. Water was distilled and deionized using Millipore Milli-RO 10 Plus and Milli-Q UF Plus (Bedford, MA) to a final resistivity of 18 MΩ. Simulated tear fluid (STF) was prepared in accordance with previously published protocols . All organic solvents used in the current study were of HPLC grade and were purchased from Sigma Aldrich Canada.
Preparation of NP-laden hydrogels
MC was initially dissolved in half of the volume of deionized (DI) water that is required for the sample under investigation. For instance, if the final concentration of MC that was required in the sample was 10 g/L, 10 g of MC was first dissolved in 500 mL of DI water. The DI water was close to boiling (~ 90 °C) when it was added to MC, and the mixture was agitated until the solids were completely immersed. The lower critical solution temperature (LCST) of MC in water ranges between 40 and 50 °C, which implies that MC is not soluble in water at temperatures above its LCST. The remainder of the water, which was at ambient temperature, was added to the aforementioned MC solution as it was stirring. The solution temperature was then rapidly lowered to 0 °C and agitation continued for an additional 15 min. HA powder was then added to the MC solution, and the mixture was stirred for another 10 min. Finally, the NP suspension was added to the hydrogel solution to yield a final concentration of 10 wt.%. This mixture was then incubated overnight at 4 °C prior to experimentation.
An Anton Paar MCR-501 rheometer equipped with a cone-and-plate geometry having a cone angle and diameter of 4° and 25 mm, respectively, was used to assess the rheology of the formulations. The edges of loaded samples were also coated with low-viscosity silicon oil in order to minimize dehydration. Strain and amplitude sweep tests were performed prior to rheological testing in order to identify the ranges of strain and frequency associated with the linear viscoelastic regime. The temperature for sol-gel transition of the formulations was identified by performing small amplitude oscillatory shear (SAOS) experiments by monitoring the crossover of storage (G′) and loss (G″) moduli at various temperatures . The temperature was increased by a rate of 1 °C/min, while the frequency was set at 1 Hz. The shear-thinning behavior of the formulations was investigated by measuring their viscosities as a function of shear rate, which ranged between 0.01 and 100 s−1. Temperature sweeps were performed on three distinct samples of the same composition in order to confirm reproducibility of the results.
Factorial design of experiments for optimization of hydrogel composition
Coded levels and actual values of the factors investigated in the factorial experiment
Coded levels of factors
Actual values of factors
HA concentration (wt.%)
MC concentration (wt.%)
NP size (nm)
The sol-gel transition temperature, which is the dependent variable, was measured using a cone and plate rheometer as described previously. The function fac.design() embedded in the DoE.base package of R was used to generate a random order of runs for the 27 experiments. Each experiment was conducted three times, and the effects and interactions between the independent variables were also obtained using R. The significance of the variable effects as well as their interaction effects was evaluated by analysis of variance (ANOVA) for each parameter. Results with a P value less than 0.5 were deemed to be statistically significant. Unlike conventional factorial designs, which facilitate identification of an optimal output, we sought to identify an output—not necessarily an optimum point—with a specific characteristic, namely a sol-gel transition temperature of ~ 32 °C. Since this output is not an optimum point, there are many possible combinations that can yield the desired characteristic. The impact of each variable on the output was also predicted using R in order to identify a formulation that gels at 32 °C. Furthermore, some of the 27 experiments were repeated (three replicates) using poly(ethylene glycol) (PEG) NPs. We observed that the composition of the NP did not exert a statistically significant influence on the sol-gel transition temperature. The results of the factorial design informed the optimization of the methodology for synthesis of the drug-loaded PEO-b-PLA NPs.
Biosynthesis of CBGA
We genetically engineered the bacterium E. coli to synthesize CBGA. Genes encoding the rate-limiting enzymes of the native non-mevalonate pathway—dxs, ispD, ispF, and idi—were overexpressed on a medium-copy pTrc plasmid under the control of an isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible trc promoter. A strong ribosomal-binding site was employed to drive translation of the transcripts and the plasmid also bears a chloramphenicol selection marker. This plasmid is hereinafter referred to as pTrc-MEP. Overexpression of the aforementioned enzymes enhances production of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which condense together to form geranyl pyrophosphate (GPP). The enzyme GPP synthase (GPPS) catalyzes the condensation of IPP with DMAPP.
In the marijuana plant Cannabis sativa, GPP then combines with OA to form CBGA. This reaction is catalyzed by an aromatic prenyltransferase called cannabigerolic acid synthase (CBGAS) . The gene expressing CBGAS  was purchased from DNA 2.0, and its codons were optimized for expression in E. coli. The synthetic construct was supplied on a high-copy plasmid whereon an IPTG-inducible T5 promoter-controlled transcription. We refer to this plasmid as pT5-CBGAS. We also separately cloned the gene for CBGAS as well as an operon comprising the genes that express GPPS and CBGAS onto a low-copy pBAD33 plasmid under the control of an arabinose-inducible promoter. These plasmids are referred to as pAra-CBGAS and pAra-GPPS-CBGAS, respectively, and both bear the chloramphenicol selection marker. Transformants bearing pT5-CBGAS were selected using kanamycin. All the plasmids used in this study are compatible with one another in co-transformed cells and neither IPTG nor arabinose were observed to affect the performance of the cognate promoters of one another in the ranges of concentrations that were evaluated in this study.
We initially transformed E. coli BL21 with pT5-CBGAS and confirmed the synthesis of the desired geometric isomer in vitro using total extracted protein. Briefly, the transformed cells were cultured in 3 mL of LB medium at 37 °C until they reached an optical density (OD) of 0.6. Protein production was subsequently induced using 1 mM of IPTG. The induced cultures were maintained at 30 °C for 4 h. The cultures were then harvested in order to prepare cell extracts for in vitro evaluation of enzyme activity. Replicates of the protein extracts were prepared from three distinct culture tubes. Each 300 μL in vitro reaction comprised 0.2 mM OA, 0.4 or 1 mM GPP, 5 mM MgCl2 and 64 μg of unpurified protein in a Tris-HCl buffer at pH 8.5. The reactions were allowed to proceed for 1 h at 30 °C. Following completion of the reactions, the metabolites were extracted in ethyl acetate and analyzed using reversed-phase HPLC on Perkin Elmer Flexar instrument. The HPLC was equipped with a Waters Atlantis C18 column (5 μm, 4.6 mm × 250 mm), and CBGA was detected using UV light at 270 nm. An isocratic mobile phase of water:acetonitrile 15:85 and 0.01% trifluoroacetic acid (TFA) at a constant flow rate of 1 mL/min and temperature of 40 °C was used. The peak was measured at a wavelength of 270 nm. For an injection volume of 10 μL, the retention time was ~ 9 min.
The concentration of GPP was deliberately varied to confirm the hypothesis that a stoichiometric excess of GPP is required for the synthesis of CBGA. Synthesis of the E-isomer of CBGA was confirmed by 1H-NMR. E. coli was then co-transformed with pTrc-MEP, and either pT5-CBGAS or pAra-CBGAS or pAra-GPPS-CBGAS and the production of CBGA were evaluated in vivo. This experiment was replicated three times. In each instance, 5 mL cultures were propagated until they reached an OD of 0.6. The cultures were then induced with 10 mM arabinose and 0.05 mM IPTG and fed with 0.5 and 0.1 mM of GPP and OA, respectively. The cultures were grown overnight for 30 °C, after which the fermentation broth was separated from the cell mass by filtration. CBGA production was then quantified through HPLC. Fifty-milliliter-scale production of CBGA for use in the formulations was subsequently undertaken for 3 days using the most productive of the co-transformed E. coli strains, and CBGA was purified from the fermentation broth using flash chromatography. Briefly, the fermentation broth was sonicated to lyse the cells and the liquid medium was filtered to eliminate cell debris. The filtrate was vigorously contacted with ethyl acetate and the organic extract was then dissolved in DMSO for purification using a Reveleris X2 flash chromatography unit. A water-methanol gradient was used to separate CBGA using a SRC C18 column.
Preparation and characterization of the drug-loaded PEO-b-PLA nanoparticles
The CBGA-loaded PEO-b-PLA NPs were synthesized by nanoprecipitation [27, 28]. Briefly, 10 mg of CBGA and 50 mg of PEO-b-PLA were dissolved in either 5 mL or 10 mL of ethyl acetate. The mixtures were then either sonicated and added dropwise to 100 mL of rapidly stirred water at room temperature or added to water as is, and the solutions were left uncovered for 3 h to facilitate evaporation of ethyl acetate and NP formation. The NPs were then extracted by ultrafiltration using Amicon Ultra-15 centrifuge filters. The filters have a MW cut-off of 30 kDa and were centrifuged at 4000×g for 8 min. The concentrated NP suspension was then washed with water and re-centrifuged.
The particle size and polydispersity index of the drug-loaded NPs were measured with a Malvern Zetasizer Nano ZS at 25 °C and at a scattering angle of 90°. Morphological evaluation was performed using a Hitachi S-3000N Scanning Electron Microscope. Briefly, a small volume of the nanoparticle suspension was placed onto a bare aluminum stub and allowed to air dry. The sample was then sputter-coated with 5 nm of gold-palladium alloy. A working distance of 5.0 mm and an accelerating voltage of 5.00 kV were used to image the sample. The NP size distribution and sphericity was also confirmed by estimating the size of 20 randomly selected particles in micrographs of six different regions within the samples using ImageJ. The encapsulation efficiency of the NPs was determined through HPLC quantification of sonicated samples of 1 g of dried NPs in 1 mL of cold acetonitrile in triplicate, as described previously .
Evaluation of the texture of the NP-hydrogel composite
The texture of the NP-laden hydrogel was surveyed using atomic force microscopy (AFM). Measurements were made using a Vecco Multimode 8 Scanning Probe Microscope. Ten microliters of the formulation was spotted on freshly cleaved mica and spread over the surface to form a thin film. The film was allowed to dry under air and then immediately analyzed. Images of a 5-μm-×-5-μm section were captured in AC tapping mode with a tip velocity of 9.8 μm/s, a loop gain of 8, and a scanning speed of 0.977 Hz. The experiment was repeated three times using distinct samples.
Assessment of in solutio release
Forty microliters of the formulation was injected into a dialysis cassette. The volumetric capacity of the unit is 100 μL, and the cassette was subsequently incubated in 4 L of STF at 32 °C under constant stirring. Ten microliters of the cassette’s contents were withdrawn at intervals of 6 h, and the samples were analyzed using HPLC. The dialysis was repeated three times.
Corneal penetration study
The delivery of CBGA through the cornea was assessed using freshly excised porcine eyeballs provided by the Centre of Comparative Medicine at the University of British Columbia. The use of a complete Franz diffusion cell, which is how penetration of drugs through tissue is typically evaluated, was avoided owing to the impact on corneal swelling on reproducibility of the data . The eyeballs were obtained with the eyelids intact, which maintains the integrity of the cornea, prevents the hydrogel and ocular surface from drying, and also ensures that the experiment faithfully approximates in vivo conditions. Each eyeball was placed on a concave construct made of plasticine clay, and the constructs were lined with cling film in order to prevent the eyeballs from rolling. The donor compartment of a Franz diffusion cell was subsequently placed atop the cornea and secured using cling film. Forty microliters of the formulation was injected into the donor compartment, and the same concentration of CBGA in mineral oil was used as a control. The entire setup comprising the eyeball atop the plasticine clay holder was immersed in a water bath at 32 °C. With the exception of the donor compartment and a small area surrounding it, the eyeball was entirely submerged in water. Remnants of the formulation were scrapped off the ocular surface after 4 h, and the surface was washed with STF. The cornea and leans were subsequently dissected and separately digested in 1 mL of methylene chloride at 60 °C for 4 h. The tissue samples were then centrifuged, and the CGBA content in the supernatant was analyzed using HPLC. The entire experiment was repeated three times for the experimental and control formulations.
Results and discussion
Rheological optimization of the NP-laden hydrogel
Microbial synthesis of CBGA
Glaucoma is characterized by high intraocular pressure, which progressively damages the head of the optic nerve. Justifiably, lowering the intraocular pressure has been an extensively exploited therapeutic strategy against glaucoma. Nevertheless, none of these medications remedy the damage that has already been inflicted to retinal ganglion cells that comprise the head of the optic nerve. Reversing this damage through elicitation of neuroprotective mechanisms could prove to be a valuable complementary strategy in addition to lowering intraocular pressure . In particular, activation of the cannabinoid receptors is a promising approach to elicit neuroprotection . The cannabinoid receptors are a unique class of G-protein-coupled transmembrane receptors that are principally located on the surface of neurons and immune cells . These receptors are also found in the gastrointestinal tract, liver, pancreas, adipocytes, eyes, vascular endothelia, spleen, and the lymph nodes. Activation of the cannabinoid receptors triggers downstream signaling and metabolic pathways that subsequently exert a major influence on synaptic transmission, cellular fate, including neuroprotection, and the body’s immune response. Secondary metabolites synthesized by the marijuana plant C. sativa rank among the strongest agonists of the cannabinoid receptors. These molecules are aptly referred to as cannabinoids and are essentially prenylated polyketides that are derived from fatty acid and terpenoid precursors . Presently, cannabinoids are produced commercially by directly extracting them from the marijuana plant. However, not only is the source material inconsistent in composition and quality, but its supply too is prone to seasonal and environmental variations. Chemical synthesis of these molecules is also quite challenging, partly due to isomerism. These considerations have greatly limited the use of cannabinoids as active pharmaceutical ingredients as well as additional investigations into the pharmacology of these compounds.
We speculate that production of GPP over a yet-to-be determined threshold has a detrimental effect on cell health since GPP is toxic to cells at higher concentrations . We subsequently cultivated the E. coli strain that co-expresses the pTrc-MEP and pT5-CBGAS in 50 mL cultures for 3 days to produce CBGA for use in the formulations. The average final titer of CBGA in these cultures is approximately 14 μg/mL and the compound is separated via flash chromatography. Each 50 mL batch produces enough material to synthesize in excess of 50 batches of NPs, and the cultures were re-cultivated to meet CBGA requirements as they arose.
Synthesis and characterization of the CBGA-loaded NPs
Performance of the NP-laden hydrogel
We have successfully developed a stimulus-responsive, in situ-forming, nanoparticle-laden hydrogel for controlled release of poorly bioavailable drugs such as cannabinoids into the aqueous humor of the eye. Using rheology, we systematically optimized the composition of the formulation to achieve a sol-gel transition temperature of 31.5 °C, which is approximately the temperature of the ocular surface. The formulation comprises 1.5 and 2.5 wt.% HA and MC, respectively, and includes CBGA-loaded PEO-b-PLA NPs having an average diameter and polydispersity index of 186 nm and 0.118, respectively. The CBGA used in this study was synthesized using genetically engineered E. coli, and we have also established a reliable process to produce CBGA-loaded PEO-b-PLA NPs via nanoprecipitation. HA and MC are biocompatible, highly mucoadhesive, and approved by the FDA as GRAS. However, while HA exhibits concentration-dependent gelation and shear-thinning characteristics , gels that solely comprise HA lack structural integrity on account of the high hydrophilicity of biopolymer. On the other hand, while MC is highly viscous and gels in a temperature-dependent manner, it does not gel rapidly enough. When HA and MC are co-formulated together, they interact with one another at the molecular level and yield a material that gels rapidly and can switch its rheology between thixotropy and temperature-dependent rheopexy. Likewise, the amphiphilicity of the PEO-b-PLA NPs is well suited for transporting lipophilic molecules such as cannabinoids through the cornea. Moreover, the size and uniform spherical morphology of the NPs is optimal for transcorneal penetration, efficient dissolution, and reduced irritancy to the ocular tissues. We also observed that incorporating NPs of a specific size further increases the stability of the hydrogel, and this observation is consistent with that of hydrogel formulations that are used for intrathecal injections [22, 38].
When the performance of the formulation was assessed in whole eyeball experiments, we confirmed the improvement in delivery of CBGA by facilitated transport and also observed a 300% increase in transcorneal penetration over the control formulation. In fact, our study is the first of its kind to report direct drug uptake by the cornea and lens from a composite NP-hydrogel vehicle. Nevertheless, there is considerable room for improvement. Only ~ 0.015% of the original CBGA load was transported through the cornea, largely due to the absence of lachrymal drainage. In fact, although the formulation was optimized to dissolve within 8 h under constant lachrymal drainage—and in solutio release studies confirmed that the bulk formulation dissolves completely within the first 3 h and sustains release of CBGA into the bulk phase over a 24-h period—we observed that very little of the formulation had dissolved after 4 h when it was applied onto excised porcine eyeballs. The formulation needs to be assessed in vivo in order to conclusively determine its efficacy in delivering molecules across the cornea and optimize its performance further, including attributes such as optical clarity of the lens. However, the formulation described herein is an excellent starting point and it switches between shear thinning and temperature thickening as intended when it is tested in a rheometer under conditions that are representative of the ocular surface. This result suggests that the formulation could coat the corneal surface by blinking, which implies that the medication could be applied as an eye drop immediately prior to the patient’s bedtime. Liquids are also more desirable than hydrogels since they are easier to manufacture at scale. Our work paves the way for the introduction of novel products targeting ocular diseases to the market.
The authors would like to thank Rishi Somvanshi, John Jackson, Fahimeh Hosseini, and Tim Hollenberg for their assistance with the experimentation.
Compliance with ethical standards
Conflict of interest
Sazzad Hossain is the Chief Scientific Officer of InMed Pharmaceuticals Inc., a biopharmaceutical company that is commercializing an anti-glaucoma treatment that employs the formulation described in this study.
- 1.Riordan-Eva P. Anatomy & embryology of the eye. In: Riordan-Eva P, Cunningham ET, editors. Gen Ophthalmol. 18th ed. New York, NY: McGraw-Hill; 1999. p. 1–26.Google Scholar
- 4.Optic neuropathy—pipeline review. London, UK; 2017.Google Scholar
- 10.Durán-Lobato M, Martín-Banderas L, Lopes R, Gonçalves LMD, Fernández-Arévalo M, Almeida AJ. Lipid nanoparticles as an emerging platform for cannabinoid delivery: physicochemical optimization and biocompatibility. Drug Dev Ind Pharm. 2015;9045:1–9.Google Scholar
- 12.Hingorani T, Gul W, Elsohly M, Repka MA, Majumdar S. Effect of ion pairing on in vitro transcorneal permeability of a Δ(9)-tetrahydrocannabinol prodrug: potential in glaucoma therapy. J Pharm Sci Elsevier Masson SAS. 2012;101:616–26.Google Scholar
- 19.Chittiboyina A, Khan I. Short synthesis of olivetolic acid via directed ortho-metalation. Planta Med. 2012;78:P42.Google Scholar
- 21.Goodwin J, Hughes RW. Rheology for chemists: an introduction. Royal Society of Chemistry. 2000;Google Scholar
- 25.Page J, Boubakir Z. Aromatic prenyltransferase from cannabis. 2011.Google Scholar
- 26.Page J, Boubakir Z. Aromatic prenyltransferase from cannabis. 2009.Google Scholar
- 29.Hernan Perez dela Ossa D, Ligresti A, Gil-Alegre ME, Aberturas MR, Molpeceres J, Di Marzo V, et al. Poly-ε-caprolactone microspheres as a drug delivery system for cannabinoid administration: development, characterization and in vitro evaluation of their antitumoral efficacy. J Control Release Elsevier BV. 2012;161:927–32.CrossRefGoogle Scholar
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