Polymeric curcumin nanoparticles by a facile in situ method for macrophage targeted delivery

Abstract Targeting macrophages is a promising strategy for improved therapy of intracellular infections as macrophages exhibit rapid phagocytosis of particles >200 nm. Entrapment of Curcumin (CUR) in nanocarriers could provide bioenhancement and macrophage targeting. We present a simple and facile in situ nanoprecipitation approach for instantaneous and on‐site generation of curcumin nanoparticles (ISCurNP). ISCurNP optimised by Box‐Behnken design exhibited average size of 208.25 ± 7.55 nm and entrapment efficiency of 90.16 ± 1.17%. Differential scanning calorimetry and X‐Ray diffraction confirmed amorphization of CUR in ISCurNP. Sustained release was observed over 72 hr in vitro at lysosomal pH 4.5. Rapid and high uptake in RAW 264.7 macrophages was confirmed by flow cytometry and high performance liquid chromatography. Confocal microscopy established localisation of ISCurNP in lysosomal compartment. The facile in situ nanoprecipitation method provides simple, scalable technology to enable macrophage targeted delivery of CUR, with great promise for improved therapy of intracellular infections.


| INTRODUCTION
Curcumin (CUR), a natural polyphenol has generated tremendous excitement as a nutraceutical due to its multifarious pharmacological activities. 1,2 The anticancer 3-6 and anti-inflammatory 7,8 properties of CUR have been widely explored. CUR, an established anti-infective for eons, is currently being extensively researched for antibacterial including antimycobacterial, antiviral, and antifungal activity. [9][10][11] Despite the excellent therapeutic potential, a considerable impediment in exploitation of CUR as a therapeutic agent is its hydrophobic nature and consequently poor aqueous solubility. 12 Nanonization of particles has proven to be a competent approach to enhance solubility and bioavailability of hydrophobic poorly soluble drugs. 13,14 Furthermore, such drugs when entrapped in nanocarriers with size titrated in the range of 200-600 nm can aid targeting to the reticuloendothelial system (RES) organs and cells, especially macrophages, the primary site of various intracellular infections. [15][16][17][18] Macrophages constitute defense system of the body and exhibit rapid phagocytosis of particles >200 nm. Targeting the macrophages by nanocarriers thus provides a viable approach to restrict growth and survival of the organisms.
Conventional nanotechnology approaches are based on multiple steps and dictate the need for sophisticated equipment. Furthermore, isolation of nanoparticles (NPs) prepared and their stabilization by freeze drying also pose significant scale-up challenges, constituting major hurdles in the development and translation of nano drug delivery systems. 19 In contrast, in situ nanotechnology presents a method wherein generation of NPs is achieved by simple addition of a monophasic preconcentrate comprising drug, carrier (polymer/lipid), and stabilizers to an aqueous phase. Our group has reported in situ nanotechnology as a facile method for instantaneous and on-site generation of NPs to enable point of care application. [20][21][22] A distinct advantage of in situ technology is total bypass of the technological hurdles and stability issues associated with conventional NP fabrication methods. First generation in situ nanotechnology relies on dilution of monophasic preconcentrates with an aqueous media like potable drinking water just prior to oral administration or dextrose/ dextrose saline injection just prior to intravenous injection. Enhanced oral bioavailability was demonstrated from a self nanoprecipitating preconcentrate of tamoxifen citrate, which on dilution with aqueous media resulted in the formation of polymeric tamoxifen NPs with 85% entrapment efficiency and average particle size <250 nm. 20 An in situ hybrid nanoparticulate drug delivery system of nevirapine designed for simultaneous targeting of multiple reservoirs in HIV exhibited >90% drug entrapment in a mixture of solid lipid NPs and micelles which was instantaneously generated when the preconcentrate was diluted with isotonic dextrose injection. Following intravenous administration in Sprague Dawley rats, the heterogenous nanosystem revealed high drug accumulation in the RES organs of liver and spleen and also the brain, which represents a remote HIV reservoir. 21 Second generation in situ nanotechnology can additionally enable passive targeting through stealth or active targeting enabled by inclusion of a receptor ligand. Herein, the aqueous phase is provided along with the preconcentrate and comprises the stealth agent or receptor ligand. An in situ primaquine nanocarboplex anchored with the asialoglycoprotein receptor ligand pullulan was successfully designed for targeting to hepatocytes. The nanocarboplex revealed an average size of~250 nm with >70% complexation. Active targeting to hepatocytes was confirmed by enhanced hepatocyte accumulation coupled with a very high hepatocytes: parenchymal cells ratio of~75: 25. 22 In the present study, first generation in situ nanotechnology approach has been exploited as a practical approach for the design of in situ polymeric curcumin NPs (ISCurNP) with the following objectives: (a) Optimization of stable and reproducible ISCurNP by the facile in situ method (b) Evaluation of macrophage uptake and subcellular localization of ISCurNP in the RAW 264.7 murine macrophage cell line, as a first step in exploiting ISCurNP as a targeted nano delivery system for intracellular infections.

| ISCurNP
Preconcentrates were added to filtered distilled water for instantaneous formation ISCurNP.

| Preliminary screening
Preliminary screening was carried out using the one variable at a time (OVAT) approach. Preconcentrates with varying amount of Kolliphor P188/Soluplus ® , PLGA, and DMA were prepared. ISCurNP was generated by adding the preconcentrate to filtered distilled water and the critical variables and working concentration range influencing formation of ISCurNP were arrived at.  Information Table S1). Experimental design and analysis was performed using Minitab 17 (licensed version). A total of 15 runs were generated by the software. All batches were prepared in triplicate and the responses were recorded to generate the polynomial equations and response surface graphs. Three test batches were generated using the desirability function and the dependent variables were predicted.

| Design of experiment: Box-Behnken design
The actual data for the predicted batches was obtained through experimentation. Closeness of the predicted data was compared with the experimental values obtained by applying t-test.
2.5 | Evaluation of preconcentrate 2.5.1 | Clarity Preconcentrate was visually evaluated against light and dark background for presence of foreign matter or undissolved components. The RP HPLC method was validated for linearity, accuracy, and precision.

| Stability
Preconcentrate was evaluated for stability as per International Conference on Harmonization (ICH) guidelines. Preconcentrate was packed in amber colored glass vials flushed with nitrogen and stored at 30 AE 2 C/65 AE 5% RH and 40 AE 2 C/75 AE 5% RH. At prescribed time intervals, preconcentrate was evaluated for drug content.
At the same time intervals, ISCurNP was generated from the preconcentrate as described in 2.3.2 and evaluated for particle size and entrapment efficiency as described below.

| Evaluation of ISCurNP
The preconcentrate was diluted with aqueous medium to generate ISCurNP which were evaluated as below: 2.6.1 | Particle size and zeta potential Particle size (PS), polydispersity index (PDI) and zeta potential measurements of ISCurNP were evaluated on Nano Brook 90 Plus PALS (Brookhaven Instruments, NY, USA). Size distribution was determined by dynamic light scattering (DLS) at 25 C. Zeta potential measurements were carried out using phase analytical light scattering (PALS). ISCurNP was suitably diluted with filtered distilled water and measurements were recorded in triplicate.

| Entrapment efficiency
The ISCurNP generated was centrifuged at 16,350 rcf for 20 min at 20 C. Aliquot (1 ml) was carefully withdrawn from the supernatant, diluted to 10 ml with methanol and CUR concentration was estimated by UV spectrophotometry at λ max 425 nm. EE was calculated using the equation below: x 100

Scanning electron microscopy
For scanning electron microscopy (SEM) analysis, a drop of ISCurNP was placed on a piece of aluminium foil mounted on carbon tube and air dried. Prior to analysis, sample was sputtered with platinum using an auto fine coater. Samples were analyzed using FEI Quanta 200 SEM with EDS.

Transmission electron microscopy
Transmission electron microscopy (TEM) analysis was performed using TECNAI 12 BT/FEI TEM at 120 kV. Briefly, a drop of ISCurNP was placed on carbon grid (Ted Pella, Inc., Redding) and air-dried followed by negative staining with 2% uranyl acetate.
Following size and entrapment evaluation, ISCurNP dispersion was centrifuged, the pellet washed and redispersed in filtered distilled water and lyophilized using LABCONCO freeze dryer (FreeZone 4.5).
This freeze-dried solid sample was utilized for Differential scanning calorimetry (DSC), powder X-ray diffraction (XRD) and Fouriertransform infrared spectroscopy (FTIR) evaluation.

| Statistical analysis
All experimental results are expressed as mean AE standard deviation of minimum three independent measurements. Student's t-test was applied for statistical analysis and p < .05 has been considered as statistically significant.  Figure S1).

| Fabrication and optimization of nanoparticles
Design of experiment-based approaches can assess single variables as well as interactions between different variables, enabling optimization with fewer experiments, resulting in improved efficiency. [27][28][29] Two-level designs like full or fractional factorial design provide restricted statistical outputs. Among the response surface designs, which are based on more than two-factor levels, the most efficient and widely used design is the three-level Box-Behnken design. [30][31][32] In this design, a midpoint of each side of a multidimensional cube is selected and set of points around these are chosen whereby extreme values are avoided. Furthermore, interactions between the independent variables are also accounted for.
The surface plots ( Figure 1)   Three test compositions ISCurNP1, ISCurNP2, and ISCurNP3 were generated using the design and the dependent variables predicted (  Amorphization of highly insoluble drugs like CUR is one strategy to enhance bioavailability. Absence of sharp endothermic peak of CUR at 183 C, in the DSC thermogram indicated conversion of crystalline CUR to amorphous form (Supporting Information Figure S2).
The disappearance of the peaks characteristic of crystalline CUR in the XRD spectra also confirmed amorphization of CUR (Supporting Information Figure S3).
FTIR spectra of ISCurNP (Supporting Information Figure S4) depicted presence of C=O stretch at 1,625 and 1,762 cm −1 NPs, due to their small size, large surface area, and surface properties are prone to aggregation. Although there was good stability up to 24 hr as indicated by PS, small aggregates were seen after 6 hr. Nevertheless, these aggregates could be readily redispersed by mild shaking and revealed good stability when monitored on PS analyzer, suggesting formation of floccules. As ISCurNP is designed for immediate administration, even 6 hr stability was considered more than adequate.
During the optimization study, dilution volume was maintained constant at 10 ml. When injected, dilution would be carried out by trained health professionals and hence a dilution volume may be prescribed. However, when administered orally the dilution would be carried out by untrained users. To understand the ruggedness under such conditions of use, we evaluated the effect of varying the dilution volume on PS and EE (Supporting Information Figure S5). ISCurNP revealed no significant change in PS and EE when the dilution volume was varied from 10 ml (two teaspoons) to 100 ml (about half a glassful; p > .05). At 5 ml faster aggregation was seen. However, a 3 hr stability was observed which was considered satisfactory. At >100 ml, a significant decrease in PS was observed (p < .05). Although considered rugged to dilution, as a consideration in translation a measuring cup of about 50-75 ml may be provided to ensure dilution volume within the desirable range.

In vitro release
In vitro release of ISCurNP was studied at pH 1.2 and pH 6.8 to mimic the pH in the stomach and intestine, respectively. At pH 1. suggesting slow but near complete release (Figure 3). Model fitting revealed CUR release from ISCurNP followed Higuchi model at both pH 4.5 and 6.8 (Supporting Information Table S5).

Stability in culture medium
Biological media components could adsorb onto NPs to alter their surface characteristics and lead to aggregation. Such aggregated NPs can alter the cellular uptake, cytotoxicity or even the in vivo fate. 36 Physical stability (size) of NPs in cell culture medium was evaluated to ensure that they do not aggregate and remain discrete when evaluated in cell lines. No significant change in PS was seen upto 48 hr (p > .05). At 72 hr, although the size increased, the average size of <400 nm was considered acceptable (Supporting Information Figure S7).

Cell viability assay
MTT assay is widely used for assessing cytotoxicity in cell lines. MTT

| Macrophage uptake
Macrophages rapidly engulf and internalize all foreign invasions including organisms and particles by phagocytosis. 38 Typically, hydrophobic particles are taken up by this pathway. 39,40 Phagocytosis results in trapping of the foreign invasion in a phagosome which coalesce with the lysosomes present in the macrophages to form phagolysosomes. The harsh phagolysosomal environment which has a low pH and lysosomal enzymes results in degradation/killing of the internalized organism. However, organisms that have mastered the art of phagosomal survival can competently arrest early phagosome maturation and hinder phagolysosome fusion. 41 The phagosome provides a safe sanctuary for such organisms. Given time, some of these organisms can also generate a protective resistant shield as in case of Mycobacterium tuberculosis. 42 Elimination of such intracellular infections poses significant challenges dictating the need for intracellular delivery of the drug at therapeutic concentration. 18,43,44 Nanocarrierbased delivery is an effective strategy for high intracellular delivery, nevertheless yet another challenge is to ensure that the drug from the nanocarrier is released out of the phagolysosome into the cytoplasmic compartment which harbors the organisms. In the present study, we monitored ISCurNP uptake and also quantified CUR concentration in the RAW 264.7 macrophage cell line.
Flow cytometry analysis relied on fluorescence emission of CUR.
The concentration and time dependent uptake of CUR is depicted in Figure 5. Both free CUR and ISCurNP revealed time dependent increase in uptake at both concentrations (p < .05). Significant uptake at 1 hr confirmed rapid internalization of ISCurNP. Further, uptake exhibited by ISCurNP was significantly higher than free CUR (p < .05).
At 3 hr, while increase in CUR concentration from 10 to 20 μM revealed approximate twofold increase, this increase was approximately threefold when the ISCurNP concentration was similarly increased.
Uptake of ISCurNP by macrophages was also monitored by confocal microscopy to understand intracellular trafficking ( Figure 6). As results confirmed 93% co-localization with the lysosomal markers. 45 We monitored the subcellular localization of NPs using Lyso Tracker™ Red and DAPI which localize in the lysosomes and nucleus respectively ( Figure 7). Our study was in concurrence with results of Kalluru

| CONCLUSION
In situ nanoprecipitation presents a facile approach for development of polymeric curcumin NPs. The low cytotoxicity and high uptake of ISCurNP in the RAW 264.7 cells confirms macrophage targeting and potential application in the therapy of intracellular infections. Based on technology that is simple and scalable, ISCurNP presents a targeted nanocarrier system with ease of translation from bench to clinic.