Spectroscopic methods can be used to assess curcumin encapsulation efficiency and oxidation state

On dilution in dimethyl sulfoxide (DMSO) curcumin had an absorbance peak at 435 nm (Fig. 1C) and molar extinction coefficient (Fig. 1D,E) of 58547 L.mol−1.cm−1, comparable to previously reported values58. The absorbance of curcumin diluted in DMSO at 435 nm obeyed Beer-Lambert’s law up to 42 µM and TPGS/Pluronic F127 nanocarriers in the absence of curcumin had no measurable absorbance at this wavelength. Spectroscopic assessment was used to determine the encapsulation efficiency (EE%) of curcumin-containing formulations after separation of unencapsulated material by 0.22 µm filtration. Spectroscopic measurements of EE% were confirmed using an established HPLC technique (Fig. 1F) with both techniques showed good agreement (4.31 ± 0.18 mg/mL versus 4.32 ± 0.33 mg/mL respectively).

Figure 1 Spectroscopic determination of curcumin content of nanocarrier formulations. (A) The keto- and enol- forms of curcumin (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione). (B) Suspensions of 4.5 mg/mL of curcumin in (left) PBS, (centre) PBS after 0.2 µm filtration to remove insoluble material and (right) in TPGS/Pluronic F127 nanoparticles after 0.2 µm filtration to remove insoluble material. (C) On dissolution in DMSO, 22 µM curcumin possesses an absorption peak of 435 nm. (D) Determination of the molar extinction coefficient of curcumin in a DMSO solvent (58,547 L.mol−1.cm−1) and demonstration that this accelerated oxidative degradation of curcumin at low pH92 results in a reduction in this molar extinction coefficient (2133 L.mol−1.cm−1 after 72 h incubation in 1 M sodium hydroxide solution) suggesting that spectroscopic assessment can be used to monitor both the encapsulation of curcumin and its degradation. (E) Dissolution of 5 mg/mL curcumin in 1 M sodium hydroxide solution (right) resulting in a rapid colour change compared to curcumin loaded nanocarriers (left). (F) Standard curve of known concentration of curcumin measured by HPLC. Full size image

Spectroscopic determination of curcumin concentration in nanocarriers can also be used to give indication of the extent of curcumin degradation. Curcumin undergoes keto-enol tautomerization (Fig. 1A), existing in the more stable keto form under acidic or neutral conditions and the more water soluble enol-form under alkaline conditions. In common with other molecules that undergo keto-enol tautomerization59, the enol form of curcumin is more prone to hydrolytic degradation60. Acceleration of curcumin degradation processes by dissolution in an alkaline buffer58, gave rise to a dramatically reduced curcumin molar extinction coefficient at 435 nm compared to formulated curcumin (Fig. 1C,D, 2133 L.mol−1.cm−1 after 72 h incubation in the presence of 1 M sodium hydroxide solution). Furthermore, incubation of CNs in alkaline conditions induced a dramatic colour change from orange to brown (Fig. 1E). Spectroscopic assessment of curcumin concentration after dissolution in sodium hydroxide indicates that the curcumin molar extinction coefficient rapidly diminished, suggesting that this technique can not only be used to assess curcumin entrapment efficiency but also be used to monitor the extent of degradation of curcumin containing formulations.

TPGS/Pluronic F127 nanocarriers enhance curcumin solubility and stability

Initially, curcumin loaded nanocarriers were prepared by incorporation it into TPGS nanocarriers. TPGS was chosen due to the low critical micelle concentration of this excipient (0.02% w/w)48, the endogenous nature and antioxidant properties of the α-tocopherol component61 and P-glycoprotein antagonism49, which enhances the barrier crossing ability of formulations containing this agent62. TPGS is present in existing ophthalmic formulations62 and both curcumin and TPGS can be readily solubilized in ethanol, a solvent which is present at concentrations of 0.8% in commercially available eye drop formulations (i.e. Optrex ActiMist 2in1 Eye Spray for Dry Irritated Eyes) so reducing risks associated with residual solvents from the manufacturing process. Furthermore, as the use of TPGS to enhance the bioavailability of orally administered drugs is well documented60. This, in combination with recent interest in the use of Pluronic F127 food-research applications63 may suggest that the novel curcumin formulation described herein may also be suitable for oral administration.

Formulation of curcumin with TPGS micelles was found to produce nanocarriers with 16 nm diameter as determined by dynamic light scattering (data not shown). Unfortunately, these formulations rapidly aggregated at 25 °C, resulting in the formation of sediment within hours of resuspension which may be indicative of Ostwald ripening processes64. Stabilisation of curcumin loaded TPGS nanocarriers was achieved by the addition of the polymeric stabiliser Pluronic F127 (a triblock copolymer of polyoxyethylene and polyoxypropylene), which has previously been used to sterically stabilise nanocarriers against aggregation51.

Curcumin-loaded nanocarriers (CN) were prepared according to the methods described, with encapsulation efficiency and average particle size determined (Fig. 2). On resuspension in PBS (pH 7.4) or HEPES trehalose buffer (pH 7.4), nanocarriers were found to encapsulate 96.0% ± 2.0% (4.32 mg/mL) and 94.2% ± 4.1% (4.31 mg/mL) of curcumin respectively. Transmission electron microscopy revealed that nanocarriers were typically 20 nm in diameter and of uniform size (Fig. 2A). These results were confirmed by dynamic light scattering (Fig. 2B,C) which identified a homogeneous particle dispersion with a z-average diameter between 16 and 20 nm suggestive of a micellar formulation.

Figure 2 Characterization of curcumin loaded nanoparticles and stability assessment over time. (A) Transmission Electron Micrograph of curcumin loaded nanoparticles (CNs) negatively stained with 1% Uranyl acetate. Scale bar = 50 nm. Dynamic light scattering revealed a homogeneous particle population which did not significantly change on storage at (B) 25 °C or (C) on lyophilization and storage at 25 °C and resuspension after 9 weeks. (D) Photograph of 1 mL lyophilized CN in 10 mM HEPES, 50 mg/mL trehalose buffer showing good cake structure. Stability studies illustrating the change in encapsulation efficiency over time when CN were stored at (E) 25 °C (solution) versus lyophilized and rehydrated. The average particle size (F) and dispersity index (G) was recorded in each case. Mean ± 95% CI. Full size image

The encapsulation efficiency and particle size of CN formulations were assessed over time after storage at 25 °C while protecting from light. The CN formulation was found to exhibit excellent stability for 9 weeks at 25 °C, with no reduction in formulation EE% (Fig. 2E), significant change in particle diameter (Fig. 2F) or dispersity (Fig. 2G) over this time. This stability study was repeated using lyophilised CNs prepared in the same buffer before storing at 25 °C while protecting from light. The residual water content calculated at 120 °C was 1.085 ± 0.050%, indicating lyophilized formulations were properly prepared. Formulations were resuspended prior to recording dispersion properties (Fig. 2E,G) which were found to remain constant and similar to those reported for liquid formulations (Table 1). EE% was found to decline by an average of 20% versus baseline at each time point assessed, suggesting this may be a result of the lyophilization or rehydration process.

Table 1 Characteristics of curcumin loaded nanocarriers and stability over time (n = 3). Full size table

Several groups have previously attempted to prepare curcumin loaded nanoparticle formulations, including; PLGA-nanocarriers38,39, solid lipid nanocarriers40,41, liposomes42,43 and exosomes44. Existing nanoparticulate formulations of curcumin possess limited stability (not assessed beyond 72 h in any study cited), only moderate curcumin loading has been achieved (<0.77 mg/mL)38 and most protocols would be difficult to translate to the clinic owing to complex, multi-step manufacture protocols requiring organic solvents. The TPGS/Pluronic F127 curcumin formulation described here compares favourably with those in the existing literature.

XRD and FT-IR spectra were acquired to ascertain the nature of curcumin once incorporated into nanocarriers (Fig. 3). The X-ray diffraction patterns of free curcumin exhibited characteristic peaks between 5° and 30°, indicative of a highly crystalline structure65. This character was lost on inclusion of curcumin in a nanocarrier formulation, indicating that curcumin has successfully been incorporated into the amorphous nanocarrier structure and is not associated with the particle surface66. FT-IR spectra reveal characteristic peaks of free curcumin at 3509 cm−1, 1626 cm−1, 1601 cm−1, 1505 cm−1, 1271 cm−1, 1024 cm−1, 948 cm−1 and 713 cm−1 which closely match previously reported values67. On incorporation into nanocarriers, the characteristic curcumin peak at 3509 cm−1 (indicative of the free hydroxyl group) merged with the broad OH peak of the TPGS/Pluronic F127 carrier at 3352 cm−1, which may suggest complex formation68. Furthermore, characteristics shifts in the aromatic C=C peak (1601 cm−1 to 1588 cm−1) and the C=O stretching, δ(CCC) and δ(CCO) in plane bending from 1505 cm−1 to 1514 cm−1 have previously been interpreted as evidence for the successful incorporation of curcumin into a complex68.

Figure 3 X-ray diffraction and FT-IR characterization of curcumin loaded nanocarrier formulations. (A) X-ray diffraction pattern of naïve curcumin (blue), empty nanocarriers (black) and curcumin nanocarriers (red). (B) FTIR analysis of (1) trehalose, (2) curcumin loaded nanoparticles, (3) free curcumin and (4) empty nanoparticles. Full size image

Formulation of curcumin into nanocarriers substantially reduced the rate of drug release compared to free drug (t 1/2 = 22.6 h versus 0.15 h respectively, Fig. 4) at 37 °C, attributed to the slow rate of release of curcumin from nanocarriers. Less than 10% of the drug was liberated after 5 h of incubation, suggesting that there was little burst release from the CN formulation. This observation supports FT-IR and XRD observations that curcumin is not merely associated with the nanocarrier surface but is localised within the hydrophobic interior in an amorphous or disordered crystalline phase, in agreement with previous work41. Together, these results suggest that the curcumin-loaded nanocarrier formulation described in this study have sustained release capability.

Figure 4 In vitro release of curcumin. In vitro release of 4.5 mg/mL curcumin from (A) 95% ethanol solution or (B) curcumin-loaded nanocarriers in PBS at 37 °C (mean ± SE, n = 3). Owing to the poor solubility of curcumin in aqueous buffers, the release of curcumin from ethanoic solutions was limited by the formation of a visible precipitate from the 0.5 h time point. No such aggregation was observed in experiments using nanocarrier curcumin. Full size image

Curcumin-loaded nanocarriers protect a retinal cell line against glutamate and hypoxia-induced injury

Glutamate excitotoxicity represents a potential mechanism leading to RGC loss in glaucoma69,70. Using an AlamarBlue cell viability assay, co-incubation of immortalised R28 cells with both CNs and empty nanoparticles was found to be significantly protective (one-way ANOVA with Tukey post-test, p < 0.001) against glutamate induced toxicity (Fig. 5A and B, IC 50 28.3 ± 3.4 mM versus 5.9 ± 1.2 mM for EM and insult only treated groups respectively, on-way ANOVA with Tukey post-test p < 0.001) with no additive effect observed on addition of curcumin to the nanoparticles (24.5 ± 1.2 mM, CN containing 20 µM curcumin). This observation is in agreement with previous studies that suggest α-tocopherol (here present in the form of TPGS) is protective against glutamate induced toxicity and this has been suggested to be a result of the anti-oxidant function of this molecule71,72. As TPGS was not also protective against cobalt chloride induced insult, this suggests curcumin and TPGS may have additive therapeutic effects.

Figure 5 Curcumin nanoparticle treatment is neuroprotective against the hypoxia mimetic cobalt chloride in immortalized retinal cells. Using an alamarBlue cell viability assay, Co-incubation of R28 cells with varying concentration of CNs significantly protected cells against (A,B) glutamate or (C,D) cobalt chloride induced insult (one-way ANOVA with Tukey post-test, ***p < 0.001). Empty nanoparticles containing TPGS were found to be neuroprotective against glutamate induced toxicity (B) but not cobalt chloride (D) induced toxicity. Full size image

Upregulation of hypoxia-related factors such as Hypoxia Inducible Factor 1α (HIF-1α) has been suggested to implicate hypoxia in glaucoma pathology73,74. Cobalt chloride (CoCl 2 ) is a hypoxia mimetic and inducer of HIF-1α75 used as an in vitro glaucoma model76. The IC 50 of R28 cells exposed to CoCl 2 for 24 h (Fig. 5C,D) was found to be significantly increased on concurrent incubation with 20 µM curcumin in the form of CN (296 ± 53 µM vs 757 ± 51 µM respectively, one-way ANOVA with Tukey post-test, p < 0.001). Treatment with an equivalent concentration of the nanoparticle in the absence of curcumin had no significant effect (296 ± 53 µM versus 354 ± 8 µM, one-way ANOVA with Tukey post-test, p > 0.05) suggesting that the protective effects observed were as a result of curcumin. Concentrations of curcumin <20 µM were not found to be neuroprotective in this model. Curcumin has previously been reported to inhibit HIF-1α in hepatocellular carcinoma cells77 and was more recently reported to supresses HIF-1α synthesis in pituitary adenomas78. HIF-1α inhibitors have previously been proposed as potential glaucoma treatment worthy of further investigation79.

Topically administered curcumin nanocarrier therapy protects RGCs in rodent models of ocular hypertension and optic nerve injury

Having established the neuroprotective activity of CNs in vitro in relation to vehicle only treatments, we next assessed the neuroprotective effects of this formulation on RGC health using an established in vivo rodent model of RGC loss. We anticipate that topically applied curcumin loaded nanoparticles will reach the retina via a combination of topical and systemic absorption routes. In support of this hypothesis, Sigurdsson et al. reported that their formulation of dexamethasone, which is a similar molecular weight to curcumin (392 versus 368 Da respectively), entered the retina 60% via topical penetration and 40% by systemic absorption route80. We anticipate that the well-documented P-gp inhibition activity of tocopherols49,81 and curcumin82, in conjunction with enhanced corneal penetration activity previously reported for PEGylated-micelle formulations83 will enhance curcumin delivery to the retina by the topical absorption route.

Optimum time points post model induction (maximal RGC loss in shortest time after induction) were chosen based on our previous work characterising the natural history of the OHT and pONT models where multiple time points were assessed after model induction57. We recently reported that administration of TPGS containing micelles did not themselves have a neuroprotective effect in vivo81, which in conjunction with our in vitro observations, suggest that any neuroprotective efficacy observed was a result of curcumin treatment. Rats received topical CNs according to the dosing regimen illustrated in Fig. 6A. Briefly, two days prior to OHT model induction, rodents began receiving two drops (35 µL each) of CNs dosed five minutes apart per day for three weeks from the date of model induction. Topical administration of CNs was found to be well-tolerated by rats with no signs of ocular irritation or inflammation reported in naive eyes monitored by a qualified ophthalmologist. The IOP profile of rodent’s after IOP elevation by injection of hypertonic saline into two episcleral veins (Fig. 6B) indicates that IOP remained significantly elevated for at least 7 days after model induction versus naive eyes. No significant difference in IOP profile between CN and OHT only groups was observed, suggesting that any neuroprotective effect of curcumin was due to IOP independent processes. RGC health was assessed histologically from whole-retinal mounts using brn3a assessment (Fig. 6C). This approach was chosen as Brn3a is a nuclear restricted and RGC specific transcription factor that exclusively label 97% of the RGC population (excluding photosensitive RGCs)84. We have also recently developed an algorithm to accurately and automatically quantify whole RGC populations in rodent models of retinopathy enabling the reliable assessment of RGC health57. Using this approach, OHT induction was found to result in a significant reduction in global RGC density of ~23% compared to contralateral eyes, which is comparable to previous studies using this model57. CN administration significantly improved the RGC density ratio between OHT eye vs contralateral untreated eyes (Kruskal-Wallis test with Dunns post test, **p < 0.01), whereas administration of un-encapsulated curcumin (FC - free curcumin) solubilised in PBS did not have this effect (Fig. 6D–F).

Figure 6 Topical curcumin nanoparticles protect RGC soma in vivo against OHT induced cell loss. (A) Schematic of in-vivo experimental design. OHT rats were randomised to no treatment or once-daily curcumin nanoparticles (CN) or free Curcumin (FC) eye-drops, beginning two days prior to elevated IOP induction. Three-weeks after surgery, animals were sacrificed and retinas flat mounted before labelling with Brn3a. RGC populations were counted as previously described57,94. Representative retinal images of comparable Brn3a labelled areas of superior retina are shown in (B) Naïve control, (C) OHT untreated and (D) OHT + CN animals. (E) All OHT animals had significantly raised IOP (mean ± SE) versus baseline until 21 days after surgery (Student T-test versus contralateral eyes **p < 0.01). There was no significant difference in IOP between OHT treatment groups at any time point suggesting any neuroprotective activity observed was IOP independent. (F) Elevated IOP in OHT only eyes was associated with a significant reduction in RGC density (~23%) in agreement with previous studies57; CN but not FC treatment, significantly reduced RGC loss (Kruskal-Wallis test with Dunns post test, **p < 0.01). Full size image

To further investigate the neuroprotective potential of topically applied CNs, whole-retinal brn3a labelled RGC population assessments were made in the pONT model (Fig. 7A). In this model, twice-daily topical administration of CNs was found be significantly protect RGCs (one-way ANOVA, ***p < 0.001). On subdivision of whole retinal mounts into superior and inferior quadrants (Fig. 7B), treatment with CNs was observed to result in preservation of RGC populations in both the superior and inferior quadrants, but this effect was more pronounced in the superior retina (two-way ANOVA, ***p < 0.001), which may imply the protective effects of curcumin therapy exert through anti-apoptotic as well as anti-oxidant mechanisms. Representative regions from the superior quadrant of Brn3a labelled retinal whole-mounts (Fig. 7C–E) illustrate RGC populations were diminished in retina subject pONT (Fig. 7D) versus naive controls (Fig. 7C). Treatment with CNs for three weeks was found to protect RGC soma from pONT induced injury (Fig. 7E). As preservation of RGC soma was observed in both the superior and inferior retinal quadrants, this suggests that curcumin may elicit neuroprotective activity via multiple pathways involving both primary and secondary neurodegeneration processes.

Figure 7 Topical curcumin nanoparticles protect RGC soma against optic nerve injury. Representative Brn3a labelled superior retinal sections taken from similar areas in (A) Naïve retina (B) pONT retina and (C) pONT model retina after daily topical CN treatment for 21 days. (D) Whole retinal RGC density measurements indicate that while pONT caused a significant reduction in RGC density, this was reduced by daily administration of CN (one-way ANOVA with Tukey post hoc test, **p < 0.01 and ***p < 0.0001). (E) Further segmentation of the each retina into superior and inferior quadrants using the method described previously57 demonstrates that topical CN prevent some RGC density loss in both the superior and inferior retina (two-way ANOVA with Tukey post hoc tests ***p < 0.001). Full size image

The possibility of TPGS mediated neuroprotection via inhibition of glutamate excitotoxicity is intriguing and may contribute to the neuroprotective effect of our formulation in vivo. In support of this hypothesis and our present in vitro findings, Nucci et al.85 previously reported that intraocular administration of a total of 10 µL of 0.5% (w/v) TPGS (equivalent to a total dose of 0.5 mg TPGS) was neuroprotective against ischemia/reperfusion injury in the rat. Previously, we reported that topical administration of TPGS at the same concentration did not have a neuroprotective effect in vivo81. This discrepancy is likely to the lower concentration reaching the retina compared to invasive application, typically estimated to be ~3% of the topically applied dose37. Although our previous work with this model suggests that administration of TPGS only did not appear to have a neuroprotective effect in its own right, a synergism between curcumin and TPGS is extremely likely, if not via the neuroprotective effects of TPGS alone, then perhaps via TPGS mediated modulation of P-gp activity, enhancing curcumin transport across ocular barriers49,81.

The neuroprotective effect of curcumin loaded nanocarriers observed in this study may be a result of treatment commencing two days before model induction, suggesting this therapy may be most effective for patients at risk of IOP spikes such as following phacoemulsification surgery86 or as a prophylactic to patients identified at high risk of developing glaucoma such as those with ocular hypertension or other glaucoma risk factors2. Furthermore, with the development of new techniques such as DARC (detection of apoptotic retinal cells) with the potential to diagnose glaucoma earlier in the disease process87, therapies to slow or prevent RGC loss at earlier stages of disease progression will play a greater role in glaucoma management.

In conclusion, this study describes a novel nanocarrier formulation of curcumin in TPGS/Pluronic F127 that increases the solubility of this poorly soluble drug by a factor of almost 400,000. This formulation incorporates 4.3 mg/mL of curcumin with an encapsulation efficiency consistently >90% and excellent stability in liquid or lyophilized forms for at least two months when stored at room temperature, as determined by HPLC and spectroscopic techniques. This formulation was found to be neuroprotective against glutamate and cobalt chloride induced injury in retinal cultures in vitro and significantly preserved RGC density in two well-established rodent models of ocular injury. In conclusion, we demonstrate that curcumin loaded nanoparticles have exciting potential for overcoming ocular barriers and may facilitate the translation of curcumin based therapies to the clinic for the treatment of ocular conditions such as glaucoma.