Liposomal-Cannabidiol Nanoformulation to Suppress HIV Replication and Reduce Oxidative Stress in Infected Microglia

Abstract

The advent of antiretroviral therapy has significantly reduced HIV-related morbidity and mortality. However, persistent HIV infection in the central nervous system continues to drive HIV-associated neurocognitive disorders (HAND). Cannabidiol (CBD), a nonpsychoactive cannabinoid with antioxidant and anti-inflammatory properties, has shown promise in clinical trials as a candidate to address cognitive impairments. Despite this potential, further research is required to elucidate CBD’s molecular mechanisms in HIV infection and to improve its brain bioavailability. To overcome these challenges, we investigated CBD’s effects on oxidative stress pathways and developed a liposomal nanoformulation (NF) to enhance its delivery and efficacy in brain cells. CBD treatment significantly upregulated APOE3 gene and protein expression while reducing HIV long terminal repeat (LTR) gene expression in infected microglia. The NF was characterized by hydrodynamic size, polydispersity index, zeta potential, encapsulation efficiency, cellular uptake, HIV infection levels, and APOE3 secretion. Successful CBD encapsulation was confirmed by liquid chromatography–mass/mass spectrometry. Importantly, the CBD-loaded NF reduced p24 antigen levels and LTR expression, increased APOE secretion, and attenuated mitochondrial reactive oxygen species production more rapidly than free CBD. This liposomal CBD NF enhances the pharmacological profile of CBD, offering a promising nanotherapeutic strategy to suppress HIV replication, reduce oxidative stress, and mitigate neurocognitive dysfunction associated with HAND.

Graphical Abstract

INTRODUCTION

Oxidative stress is a stage generated by the accumulation of reactive oxygen species (ROS) and the cells’ limited capacity to eliminate these intermediates or cause damage. The accumulation of reactive species impairs several physiological processes and triggers neuro and systemic inflammation.¹ The brain requires high levels of energy, which leads to high consumption of glucose and oxygen. Therefore, an elevated number of mitochondria exists in the brain. Consequently, the excesses of mitochondrial work and byproducts make the brain susceptible to abundant ROS. Additionally, there is a reduced expression of the key antioxidant enzymes such as SOD, GpX, a restricted entrance of them to the brain of due limited permeability across the blood-brain barrier.^2,3 Noteworthy, the presence of oxidative stress increases the risk of lipid peroxidation.^2,3 Several neurological disorders are driven by excessive production of ROS, often resulting from mitochondrial dysfunction. Notable examples include Parkinson’s disease, Alzheimer’s disease, and various age-associated neurodegenerative conditions.²

Even though HAART (Highly Active Antiretroviral Treatment) has been implemented to treat HIV infection, there is still a significant prevalence of HIV-associated neurocognitive disorders (HAND).⁴ Currently, the prevalence is a 12 to 25% prevalence of mild cognitive impairments regardless of systemic viral suppression.^4–6 HIV crosses the blood-brain barrier in early infection, where it infects and replicates within microglial cells.^5–8 Moreover, neurotoxic HIV proteins released extracellularly impair brain function and exacerbate oxidative stress.⁹ The restrictive nature of the blood-brain barrier (BBB), including limited antiretroviral penetration, makes HIV control in the CNS particularly challenging. Numerous antiretroviral drugs have been evaluated for CNS penetration, neurotoxicity, and their indirect effects on cognitive function.^4,6,10 Cognitive improvements typically emerge after one year of HAART and vary by drug type and individual factors. Notably, antiretrovirals may induce oxidative stress, highlighting the need for adjunct agents to mitigate this effect in the brain.⁸

Cannabidiol (CBD), a nonpsychoactive compound of the cannabis plant, has gained recognition as a nutraceutical for treating neurocognitive disorders.¹¹ CBD is a liposoluble compound, administered in many medical preparations as oil. Previous studies in mice showed that CBD attenuated neuroinflammation, recovered neurological functions, and improved BBB permeability.^12,13 Moreover, we recently reported that 1 μM of CBD reduced the activation of the inflammasome pathway by decreasing the activity of Caspase 1, expression of NLRP3 gene, and secreted inflammatory cytokines in human microglia cells infected with HIV-1 Bal.⁷ As an antioxidant, CBD has been shown to inhibit superoxide radical formation and activate redox-sensitive pathways, including glutathione peroxidase and reductase.^14–16 It also attenuates pulmonary arterial hypertension, restores mitochondrial function, and normalizes hypoxia-induced oxidative stress in smooth muscle cells.¹⁷ The FDA had approved Epidiolex, a CBD oil preparation to treat epilepsy.^18,19

Clinical trials have demonstrated that CBD exerts anxiolytic and cognitive-enhancing effects with minimal serious side effects. CBD has been administered orally, vaporized, intravenously, and intramuscularly, with the administration route significantly influencing cognitive efficacy. Oral delivery results in lower systemic peak concentrations¹¹ than inhalation, requiring higher doses for comparable effects.^11,20 Drug penetration across the BBB depends on lipid solubility, molecular size, and charge.^20,21 CBD’s high lipophilicity facilitates brain entry.²¹ However, BBB permeability varies with administration route, dosage, and formulation type. Despite CBD’s apparent permeability exceeding 4.0 × 10⁻⁶cm/s, formulation improvements are ongoing.¹⁹ Although side effects are rare, weight gain, drowsiness, lipid dysregulation, and hepatomegaly have been reported. Caution is advised when coadministered with barbiturates and antipyrine due to CYP3A and CYP2C inhibition.²² Multiomics analyses revealed that prolonged high-dose CBD treatment induces dose-dependent apoptosis.^23,24 Therefore, strategies enhancing absorption and bioavailability, such as lipid incorporation, optimized administration routes, and increased lipophilicity, may improve CBD’s brain efficacy.

Nanoparticles (1–100 nm) possess diverse tunable properties such as size, surface charge, shape, composition, stability, biodegradability, biocompatibility, loading capacity, tissue penetration, and release kinetics, that enhance their therapeutic potential.^25,26 Lipid-based nanoparticles are particularly valued for their biocompatibility, stability, and ability to encapsulate both hydrophilic and hydrophobic drugs.²⁷ Several FDA-approved lipid nanoformulations (NFs) are used in vaccines, cancer therapy, drug delivery, imaging, and diagnostics.²⁷ Oral lipid-based formulations improve systemic bioavailability, permeability, and lymphatic transport while bypassing first-pass metabolism.^28–30 Liposomal formulations containing egg phospholipids benefit the brain. Choline, a phospholipid, supports membrane synthesis and neurotransmitter acetylcholine production, critical for memory, mood, and BBB integrity.³¹ Cholesterol, also common in liposomes, maintains membrane integrity and modulates oxidative stress.³²

Based on these advantages and nanoparticle surface functionality, we propose a novel liposomal CBD nano formulation designed for targeted delivery to reduce HIV-1 Bal infection and oxidative stress in the brain, potentially enhancing CBD’s efficacy.

MATERIALS AND METHODS

Cell Culture.

C20 Human microglia cell line was donated by Dr. Karn’s lab. Cells were grown in DMEM medium supplemented with 10% FBS in a 37 °C incubator with 5% CO₂.³³

HIV Infection.

C20 cells were infected with HIV-1Ba-L (National Institutes of Health; AIDS Research and Reference Reagent Program), adding a dose of 100 ng of p24 units per million cells. Cells were incubated with the virus overnight, with a previous 8 h activation with 2 μg/mL Polybrene (Catalog # H9268; Sigma-Aldrich). The media was removed, and cells were extensively washed with PBS to eliminate unbound viruses.³⁴ Fresh media containing an adequate concentration of CBD (Catalog #1570; Tocris biosciences) and liposomes, and liposomes containing CBD were added according to the experimental design. Infection was maintained for 6 days, and CBD was replenished every other day. DMSO was used as a vehicle control. An equal volume of DMSO was applied to control cells not treated with CBD. Supernatants were collected to later test HIV infection by p24 ELISA (Catalog #NC9130878; Zeptometrix) and other proteins, such as APOE.

Quantitative Real-Time PCR (qRT-PCR).

Gene expressions were quantified using the real-time qRT-PCR method. Total RNAs were obtained after collecting the cell pellets from microglia cells infected with HIV and treated with 1 μM of CBD. The cell pellet from each treatment was used for the mRNA isolation using the RNA Easy Plus kit (Catalog # 7134, Qiagen). Purity and concentration of the RNA were measured by microspot RNA reader (Synergy HT Multi-Mode Microplate Reader from BioTek, US). One μg of RNA from each control and treatment was reverse transcribed, followed by real-time PCR for LTR gene (HIV LTR Pa03453409_s1; Thermofisher Scientific). GAPDH (Hs99999905_m1) was used as a housekeeping gene. Data represent the means ± standard error of three independent experiments.

RT² Profiler PCR Array Human Oxidative Stress Pathway Plus RT PCR Array.

RT² Profiler PCR Array Human Oxidative Stress Pathway Plus RT PCR Array (Catalog# PAHS-065Y; Qiagen) was done in C20 Human microglia cells (Control, vehicle control, CBD 1 μM, HIV, and HIV CBD 1 μM). The cell pellet from each treatment was used for the mRNA isolation using the RNA Easy Plus kit (Catalog # 7134, Qiagen). Purity and concentration of the RNA were measured by a microspot RNA reader (Synergy HT Multi-Mode Microplate Reader from BioTek, US). One μg of RNA from each control and treatment was used for the first-strand cDNA synthesis (Catalog # 330401, Qiagen). The genomic DNA elimination step was performed before reverse transcription. Relative abundance of each mRNA was assessed using RT2 SYBR Green/ROX PCR Master mix (Cat # 330520, Qiagen) and aliquoted in equal volumes (25 μL) to each well of the real-time PCR array plates. The real-time PCR cycling program (as indicated by the manufacturer) was run on a Stratagene Mx3000p qRT-PCR thermal cycler.

Array was performed using 96 96-well formats using Stratagene Mx3005p qRT-PCR instrument. Baseline and Threshold values of each plate corresponding to each treatment were manually adjusted following Qiagen’s recommendation. Then, Ct values were obtained using Stratagene MaxPro software. CT data were uploaded into the data analysis template on the manufacturer’s Web site, GeneGlobe (Qiagen.com). The fold-change calculations for each gene were calculated using the well-established ΔΔCT method, p-values by student’s t test (two-tail distribution and equal variances between the two samples). We selected 2 significantly dysregulated (>2 fold and p ≤ 0.05) genes to continue our study. Controls are also included on each array for genomic DNA contamination, RNA quality, and general PCR performance.

This array interrogates genes involved in the Reactive Oxygen Species (ROS) Metabolism, Superoxide Dismutase (SOD) pathway, Oxidative Stress Responsive Genes, Oxygen Transporters, and other specific genes that participate in the antioxidant cellular response. Table S1 shows the list of genes per functional category included in the array (Supporting Information).

Apolipoprotein E (APOE) ELISA.

Supernatants from treated and/or infected C20 cells were collected after 6 days of treatment to assess the concentration of extracellular APOE protein using Speedy Human APOE ELISA Kit (Catalog # SE50006; Proteintech) following the provider’s instructions. This kit detects total APOE levels, which includes all three major isoforms (APOE2, APOE3, and APOE4) and It is designed to measure the total concentration of APOE without distinguishing between the isoforms. Supernatants from cultures treated with liposomal nano formulation were assayed by ELISA to determine the expression of APOE after 3 and 6 days.

ROS Assay.

Reactive species production was tested by DCFDA (2′,7′-Dichlorofluorescin diacetate) (Thermofisher, Cat #D399). C20 cells were treated with 1 μM of CBD in the presence and absence of HIV infection. After 6 days of infection, the ROS assay was performed following the provider’s instructions. Cells were treated with Hoechst 33342 Fluorescent Staining to standardize the ROS production per number of cells. Cells treated with H₂O₂ were used as positive control. The ROS assay was measured at Ex/Em: ~ 492–495/517–527 nm using in Biotek Synergy HT multimode microplate reader instrument. Vehicle control was subtracted accordingly.

MitoROS Assay.

C20 cells were treated with 1 μM of CBD, free and contained in liposomes, in the presence and absence of HIV infection. After 6 days postinfection, the MitoRos 580 assay (catalog #16052; Aat Bioquest) was performed following the provider’s instructions. Since the nano formulation contains green fluorescence, we checked the production of ROS using this MitoRos reagent that fluorescence in the red spectrum. Cells were treated with Hoechst 33342 Fluorescent Staining to standardize the ROS production per number of cells. Cells treated with H₂O₂ were used as positive control. MitoROS 580 was used to check the developed ROS after treatments and was measured at Ex/Em = 510/580 using a Biotek Synergy HT multimode microplate reader instrument. Vehicle control was subtracted accordingly.

Preparation of Liposomal Formulation.

Liposomal nano formulations (LNFs) (Scheme 1) were prepared using the Hydration method. LNFs were prepared with and without CBD. Egg Phospholipids, Cholesterol, and 1-palmitoyl-2-(dipyrromethene boron difluoride) undecanoyl-sn-glycero-3-phosphocholine (TopFluor) were mixed wherein the lipids comprise at a molar ratio of 66.5 PC: 33 Chol: 0.5 Topfluor. (Egg Phospholipids (Catalog # 840051C, Avantis polar lipids), Cholesterol (Catalog #C8667, Sigma-Aldrich), and 1-palmitoyl-2-(dipyrromethene boron difluoride) undecanoyl-sn-glycero-3-phosphocholine (TopFluor) (Catalog # 810281P, Avantis polar lipids), CBD (Catalog #1570; Tocris biosciences)). Further, the lipid was hydrated with and without CBD, to form an unilamellar lipid film using a rotatory water bath. The lipid film was hydrated in PBS (1 mL) in a water bath at 37 °C for 1 h. Pipetting was done until the lipid film came off, followed by centrifugation at 21000g for 1 h. The supernatant is used to calculate the amount of CBD encapsulated and to estimate the encapsulation efficiency in the UV spectrophotometer (U-2910, Hitachi). OD value is extrapolated in the standard curve after blank subtraction. The pellet was redispersed in 1 mL PBS. The liposomal preparation was extruded using a 0.2 μm membrane 8 times back and forth as described by us.^35,36

Scheme 1.

Scheme of the (a) Blank Liposomal Nanoformulation and (b) CBD Liposomal Nanoformulation

Characterization of NFs.

Two different nano formulations (1-liposome only; 2-CBD liposome) were freshly prepared and used for treatments which were characterized by Zeta size, Polydisperse Index, Zeta potential, and encapsulation efficiency accordingly using Zetasizer nano series (ZS90, Malvern) and Spectrophotometer (U-2910, Hitachi), respectively. The presence of CBD in the liposome was confirmed by LC-MS. Bruker LC-MS instrument was equipped with an Acclaim 120 RSLC C18 50 × 2.1 mm; 2.2 μm HPLC column and with the mobile phases A and B (water and acetonitrile, both 0.1% formic acid). The LC-MS/MS method was set up under the following parameters: Column oven should be 40 °C. Run time is 30 min. ESI source parameters: Spray Voltage (+): 4500 V; Spray Voltage (−): 3500 V; Cone Temperature: 350 °C; Cone Gas Flow: 20; Heated Probe Temperature: 350 °C; Heated Probe Gas Flow: 30; Nebulizer Gas Flow: 30; Exhaust Gas: ON.

XTT Cell Viability Assay.

Cell cytotoxicity was assayed by the XTT assay (Cat# 30–1011K; ATCC) in Human Immortalized Microglia cells, C20 (Donated by Dr. Karn’s lab) after 6 days post HIV infection and/or treatment with CBD, Liposome containing CBD. The XTT cell viability assay was performed to confirm that NF was not toxic. We used higher concentrations of NF than therapeutic doses. The XTT assay was measured at 475 and 660 nm, using a Biotek Synergy HT multimode microplate reader instrument. Results are expressed as a percentage of cell viability compared to control cells.

Liposome Uptake by C20 Human Microglia cells.

Cells were treated with different liposome-containing CBD concentrations to reach the therapeutic concentration (1 μM). Similar volumes were used for the liposome alone as a control. After 3 and 6 days of treatment, the cells were collected, fixed, and permeabilized using the Fixation and Permeabilization Solution Kit (BD; Cat. No. 554715). Immortalized human microglial cells (C20) were used as an untreated and negative control. Cells were acquired on an Accuri C6 flow cytometer (BD Accuri; Ann Arbor, MI) and analyzed with FlowJo software (Tree Star, Inc.; Ashland, OR). Gating strategy: Forward and side scatter density plots were used to identify the interested cell population and to exclude debris. Doublets were excluded by the Forward scatter height vs forward scatter area density plot. Single-parameter histograms were used to identify FITC-positive cells, transferring the gate from untreated cells to remaining treatment variants, including HIV-infected and uninfected cells. Results were expressed as the number of positive cells.

APOE Genotyping.

To assess APOE variants in C20 cells, the genomic region encompassing two single-nucleotide polymorphisms, rs429358 (T > C) and rs7412 (C > T), was amplified and genotyped using Sanger sequencing. Briefly, genomic DNA from cultured C20 cells was isolated using the PureLink Genomic DNA kit. The region of interest was amplified using a specific pair of primers, APOE-F (5′ -GCCTACAAATCGGAACTGGAG- 3′) and APOE-R (5′ -CTCCTTCACCTCGTCCAGG- 3′), and Platinum II Taq Hot-Start DNA Polymerase using the parameters and cycling conditions recommended by the manufacturer. The amplicon size was confirmed using agarose gel electrophoresis. The PCR product was purified with the QIAGEN PCR Purification Kit and sent to Eurofins Genomics for sequencing. The APOE variants were identified by comparison of the sequence data to the APOE Reference Sequence (NG_007084.2).

Statistical Analysis.

GraphPad Prism software (Version 10.4.2; https://www.graphpad.com) was used to graph and conduct statistical analysis for all the experiments reported in this work. Outliers were identified by the ROUT method. Cleaned data was used for analysis. Normality distribution was evaluated by the Kolmogorov–Smirnov normality test. After the ANOVA was conducted, followed by Dunn’s or Tukey’s Multiple Comparison Test as posthoc tests. Experiments were performed more than three times in independent experiments. A p-value threshold of less than 0.05 (p < 0.05) was established to denote statistically significant differences. Statistical significance is indicated as * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001; **** = P ≤ 0.0001.

Our work was approved by Institutional Biosafety Committee (IBC-24–003-AM02).

RESULTS

CBD Upregulated the Expression of APOE and TRAPPC6A Genes in the Presence of HIV.

C20 cells were infected with HIV as described in the Materials and Methods section. Oxidative stress-related gene expression was assessed using the RT² PCR Array Oxidative Stress Pathway Plus. Fold regulation was calculated relative to uninfected controls and between treatment groups. Table 1 shows the most dysregulated genes compared to the control and among groups, whereas Table S2 (Supporting Information) presents their expression levels compared to the control. Our results showed that CBD in HIV infected cells upregulated the APOE (fold regulation (FR)=14.22; p = 0.044187) and TRAPPC6A (Fold regulation = 2.79; p= 0.00996) genes in a significant manner. More dysregulated genes were obtained in our experimental condition for HIV such as BNIP3 (FR = 2.24; p = 0.032138) PRDX1 (FR = 3.88; p = 0.046784), PRDX3 (FR = 2.76; p = 0.033039), SOD1 (FR = 3.74; p = 0.049385) HSPA1A (FR = −2.46; p = 0.013177), SRXN1 (FR= −2.24; p = 0.007639); TXNRD2 (FR=−2.72; p = 0.039631).

Table 1.

RT² Profiler PCR Array Human Oxidative Stress Pathway Plus^a

HIV vs CT			HIV CBD vs Ct
Gene Symbol	Fold Regulation	p value	Gene Symbol	Fold Regulation	p value
BNIP3	2.24	0.032138	APOE	3.99	0.02704
PRDX1	3.88	0.046784
PRDX3	2.76	0.033039
SOD1	3.74	0.049385
HSPA1A	−2.46	0.013177
SRXN1	−2.24	0.007639
TXNRD2	−2.72	0.039631
HIV CBD vs HIV			HIV CBD vs CBD
Gene Symbol	Fold Regulation	p value	Gene Symbol	Fold Regulation	p value
APOE	14.22	0.044187	APOE	5.16	0.016023
TRAPPC6A	2.79	0.00996

^aRT PCR Array (Catalog# PAHS-065Y; Qiagen) was done in C20 Human microglia cells (Control, vehicle control, CBD 1μM, HIV and HIV CBD 1μM). The cell pellet from each treatment was used for the mRNA isolation using the RNA easy plus kit (Catalog # 7134, Qiagen). Purity and concentration of the RNA were measured by microspot RNA reader (Synergy HT Multi-Mode Microplate Reader from BioTek, US). 1 μg of RNA was used for the first-strand cDNA synthesis (Catalog # 330401, Qiagen). The genomic DNA elimination step was performed before reverse transcription. Relative abundance of each mRNA was assessed using RT2 SYBR Green/ROX PCR Master mix (Cat # 330520, Qiagen). The real-time PCR cycling program (as indicated by the manufacturer) was run on a Stratagene Mx3000p qRT-PCR thermal cycler in 96 well format. Baseline and Threshold values of each plate corresponding to each treatment was manually adjusted following Qiagen recommendation. Then, Ct values were obtained using Stratagene MaxPro software. CT data were uploaded into the data analysis template on the manufacturer’s website GeneGlobe (Qiagen.com). The fold-change calculations for each gene were calculated using the well-established ΔΔCT method, p-values by student’s t-test (two-tail distribution and equal variances between the two samples). We selected 2 significantly dysregulated (>2 fold and p ≤ 0.05) genes to continue our study. Controls for genomic DNA contamination, RNA quality, and general PCR performance were included.

CBD Upregulated the Secreted APOE Protein in HIV Infected Cells.

Following HIV infection, C20 cells were treated with 1 μM CBD for 3 and 6 days. Supernatants were analyzed by ELISA to assess extracellular APOE protein levels. CBD treatment significantly upregulated APOE expression in HIV-infected cultures, whereas no significant change was observed in uninfected cells treated with CBD alone at 3 or 6dpi (Figure 1a, 1b). Furthermore, APOE levels were significantly higher in HIV-infected cultures treated with CBD compared to CBD-only treated cultures. No other significant changes in protein expression were detected under the conditions tested at either time point (Figure 1a, 1b).

Figure 1.

APOE protein relative expression. Results show the relative expression of the APOE protein. Supernatants from treated and/or infected C20 cells were collected after 3 (a) and 6 (b) days after treatment to assess the concentration of extracellular APOE protein using Speedy Human APOE ELISA Kit (Catalog # SE50006; Proteintech) following provider instructions. This kit detects total APOE levels, which includes all three major isoforms (APOE2, APOE3, and APOE4) and It is designed to measure the total concentration of APOE without distinguishing between the isoforms. Supernatants from cultures treated with liposomal nano formulation were assayed by ELISA to determine the expression of APOE after 6 days. All the data was analyzed using GraphPad Prism software. The normality distribution was evaluated by Kolmogorov–Smirnov test, while statistical significance was calculated by ANOVA and Dunns Multiple Comparison Test as a post hoc test. Statistical significance is indicated as * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001; **** = P ≤ 0.0001.

CBD Decreased the HIV LTR Expression While p24 and ROS Did Not Change Significantly.

Microglial cells were infected with HIV and treated with 1 μM CBD for 6 days, as described in the Materials and Methods. Cells were then harvested for analysis of LTR gene expression (Figure 2a), and culture supernatants were collected for p24 quantification via ELISA (Figure 2b). In parallel, treated cells were incubated with DCFDA to assess reactive oxygen species (ROS) levels (Figure 3). Our results demonstrated that CBD significantly reduced LTR gene expression, suggesting a potential decrease in viral transcription. In contrast, p24 levels and overall ROS production remained unchanged, although a downward trend was observed.

Figure 2.

HIV expression. a) Quantitative real time PCR (qRT-PCR). Gene expression was quantitated using real time qRT-PCR method. Total RNAs were obtained after collecting the cell pellets from microglia cells infected with HIV and treated with 1 μM of CBD. The RNAs were reversed transcribed followed by real time PCR for LTR gene (HIV LTR Pa03453409_s1; Thermofisher Scientific). GAPDH (Hs99999905_m1) was used as a housekeeping gene. b) Concentration of HIV-1 p24 antigen was assessed by p24 ELISA (Cat# 0801111; Zeptometrix, Buffalo, NY). Supernatants from cells were infected and/or treated with1 μM of CBD were used. Data represent the means ± standard error of three independent experiments. All the data was analyzed using GraphPad Prism software. The normality distribution was evaluated by Kolmogorov–Smirnov test, while statistical significance was calculated by ANOVA and Tukey’s Multiple Comparison Test as a post hoc test. Differences were considered significant at p ≤ 0.05. Statistical significance is indicated as * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001; **** = P ≤ 0.0001.

Figure 3.

General ROS detection. Results are presented as ROS ratio, DCFDA/Hoestch. Data were analyzed using GraphPad Prism 10 software. Outliers were identified by Rout method and cleaned data was analyzed. The normality distribution was evaluated by Kolmogorov–Smirnov test, while statistical significance was calculated by ANOVA followed by Dunn’s Multiple Comparison Test as a post hoc test. Differences were considered significant at p ≤ 0.05. Data represent the means ± standard error of three independent experiments. Statistical significance is indicated as * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001; **** = P ≤ 0.0001.

Liposomal Nanoformulation Physical Chemical Characterization.

Based on these initial findings, we proceeded to incorporate CBD into a liposomal nanoformulation (Scheme 1) to evaluate whether this delivery system could enhance the observed effects of CBD. The liposomes were characterized in terms of hydrodynamic size, polydispersity index (PDI), zeta potential, and encapsulation efficiency (Figure 4). The unloaded liposomes exhibited an average hydrodynamic size ranging from 161 to 370 nm, a PDI of 0.200, and a zeta potential of − 5.13 mV. Upon incorporation of CBD, the nanoformulation (NF) showed an encapsulation efficiency of 88.58% ± 8.66, a reduced hydrodynamic size ranging from 109 to 238.4 nm, a PDI of 0.187, and a zeta potential of − 1.88 mV.

Figure 4.

Characterization of liposomal nanoformulations (NFs). NF were prepared using the hydration method as detailed in the Materials and Methods section. (1) Blank liposomes and (2) CBD-loaded liposomes were freshly prepared and subjected to physicochemical characterization, including Z-average, polydispersity index (PDI), and zeta potential, using a Zetasizer Nano Series (ZS90, Malvern). Encapsulation efficiency of CBD within the liposomes was determined spectrophotometrically using a UV–Vis spectrophotometer (U-2910, Hitachi).

The presence of CBD in the formulation was confirmed by LC-MS analysis. The results showed that the method has good linearity over the concentration with R² = 0.9953 (Figure 5a). Chromatograms revealed a single, sharp peak at a retention time of 4.0 min, consistent with the standard (Figure 5a). Mass spectrum displayed a predominant ion at m/z = 315.2348, corresponding to the [M + H]+ molecular ion of the CBD ([M + H] = 315.2319+[2M+NH4] = 646.4830). Additional fragment ions observed at m/z = 316.2362 and 317.2376 supported the proposed fragmentation pattern (Figure 5b). No significant background or interfering peaks were detected in the negative controls, confirming the specificity of the CBD detection. The liposome-only sample showed a signal below the limit of detection (1 ppm) for CBD, whereas the Liposome +CBD sample tested positive, confirming successful encapsulation (Figure 5c).

Figure 5.

LC-MS characterization of CBD-liposomal Nanoformulation. The figure displays chromatograms for CBD across a concentration range of 1 to 100 μg/mL (A), samples acquired (B), and the mass spectrum (C). The chromatographic data includes representative LC areas corresponding to each CBD concentration, demonstrating the method’s sensitivity and linearity. The presence and quantification of CBD in each analyzed sample are confirmed through consistent retention times and peak intensities.

Liposomal Nanoformulation Cytotoxicity and Cell Uptake.

The resulting NFs were utilized to deliver 1 μM of CBD to microglial cells. Assessments included cytotoxicity, cellular uptake of the formulation, secreted APOE protein levels, p24 antigen levels, and LTR gene expression at two time points, 3- and 6-days post-treatment. Both the unloaded liposome and the CBD-loaded liposome exhibited no cytotoxic effects, even at concentrations exceeding the therapeutic dose. No toxicity was obtained when liposomes were used to deliver 10μM and 20μM of CBD, which required proportionally increased amounts of the liposomal carrier (Figure 6).

Figure 6.

Cytotoxicity of Liposomal NF. Cytotoxicity was assayed by the XTT assay (Cat# 30–1011K; ATCC) in Human Immortalized Microglia cells, C20 after 6 days post HIV infection and/or treatment with CBD, Liposome, Liposome containing CBD. The XTT assay was measured at 475 and 660 nm, using a Biotek Synergy HT multimode microplate reader instrument. Results are presented as percentage of control. Outliers were identified by Rout method and cleaned data was used to assess the statistical differences. The normality distribution was evaluated by Kolmogorov–Smirnov test, while statistical significance was calculated by ANOVA followed by Dunns Multiple Comparison Test as a post hoc test. Differences were considered significant at p ≤ 0.05. Statistical significance is indicated as * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001; **** = P ≤ 0.0001.

Liposome uptake after 3 days of treatment (Figure 7) was higher in uninfected cells treated with CBD-containing formulations, suggesting that CBD may facilitate the internalization of liposomes composed of phosphatidylcholine and cholesterol. In uninfected cultures, treatment with liposome-CBD increased uptake by 20%, reaching approximately 80% of total cells. In contrast, a marked reduction in liposomal uptake was observed in HIV-infected cultures. Notably, no significant difference in uptake was detected between the unloaded and CBD-loaded liposomes in infected cells; however, uptake still reached nearly 40% of total cells after 3 days.

Figure 7.

Cell uptake studies after 3 days post treatment. Cells were treated with liposome-containing CBD concentration to reach the therapeutic concentration of 1 μM. Similar volumes were used for liposome alone as a control. At 3dpi ad treatments, the cells were collected, fixed, and permeabilized. C20cells were used as an untreated and negative control. Results were expressed as numbers of positive cells. All the data was analyzed using GraphPad Prism software 10. Outliers were identified by Rout method and cleaned data was used to assess the statistical differences. The normality distribution was evaluated by Kolmogorov–Smirnov test, while statistical significance was calculated by ANOVA and Tukey’ s Multiple Comparison Test as a post hoc test. Differences were considered significant at p ≤ 0.05. Statistical significance is indicated as * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001; **** = P ≤ 0.0001.

The study was extended to 6 days postinfection, with cells retreated using either free CBD or CBD-loaded liposomes. By day 6, HIV-infected cells exhibited the highest percentage of fluorescence. In contrast, cells treated with unloaded liposomes showed only 40% fluorescence at this time point. Notably, this same group exhibited over 60% fluorescence on day 3, indicating a time-dependent decline in liposome uptake in the absence of CBD (Figure 8).

Figure 8.

Cell uptake studies after 6 days post treatment. Cells were treated with liposome-containing CBD concentration to reach the therapeutic concentration of 1 μM. Similar volumes were used for liposome alone as a control. After 6 dpi and treatments, the cells were collected, fixed, and permeabilized. A) It shows the individual histograms while B Represents the overlaying histograms and bar graph. Results were expressed as numbers of positive cells. All the data was analyzed using GraphPad Prism software 10. Outliers were identified by Rout method and cleaned data was used to assess the statistical differences. The normality distribution was evaluated by Kolmogorov–Smirnov test, while statistical significance was calculated by ANOVA and Tukey’ s Multiple Comparison Test as a post hoc test. Differences were considered significant at p ≤ 0.05. Statistical significance is indicated as * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001; **** = P ≤ 0.0001.

Liposomal Nanoformulation Enhanced the Effectiveness of CBD in Reducing HIV Infection and Modulating APOE Expression at 3 Days Postinfection.

HIV-1 p24 and LTR-HIV expression were upregulated across all treatment groups, confirming successful HIV infection throughout the experimental timeline. As shown in Figure 2a, treatment with free CBD significantly reduced LTR-HIV mRNA expression, while secreted p24 protein levels remained unchanged after 6 days of infection (Figure 2b). In contrast, when HIV-infected cultures were treated with liposome-encapsulated CBD, p24 levels were significantly reduced compared to the untreated HIV group at 3 dpi (Figure 9a). A comparable decrease in p24 was also observed when comparing free CBD to liposomal CBD, suggesting that the nano formulation enhances the antiviral efficacy of CBD. Interestingly, no significant difference was detected between the HIV liposome-only and HIV liposomal CBD groups, which may indicate an inherent antiviral effect of the CBD component within the formulation (Figure 9a). A decay in LTR-HIV transcript levels, indicative of decreased viral transcription, was observed at 3dpi in cultures treated with both unloaded and CBD-loaded liposomes compared to the HIV-only group (Figure 9b). In contrast, treatment with free CBD did not result in a significant decrease, suggesting that nano formulation enhances the antiviral effect of CBD. However, unloaded liposomes reduced LTR expression compared to HIVCBD.

Figure 9.

CBD Liposomal NF impact on HIV replication. a, c) Concentration of HIV-1 p24 antigen was assessed by p24 ELISA (Cat# 0801111; Zeptometrix, Buffalo, NY). Supernatants from cells infected and/or treated with1 μM of CBD encapsulated in a liposomal nano formulation and free form were used. b, d) Quantitative real time PCR (qRT-PCR). Gene expression was quantitated using real time qRT-PCR method. Total RNAs were obtained after collected the cell pellets from microglia cells infected with HIV and treated with 1 μM of CBD. The RNAs were reversed transcribed followed by real time PCR for LTR gene (HIV LTR Pa03453409_s1; Thermofisher Scientific). GAPDH (Hs99999905_m1) was used as a housekeeping gene. Data represent the means ± standard error of more than three independent experiments. All the data was analyzed using GraphPad Prism software version 10. Outliers were removed by Rout method and cleaned data was used to assess the statistical differences. The normality distribution was evaluated by Kolmogorov–Smirnov test, while statistical significance was calculated by ANOVA and Dunn’ s Multiple Comparison Test as a post hoc test. Differences were considered significant at p ≤ 0.05. Statistical significance is indicated as * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001; **** = P ≤ 0.0001.

Upon extending the infection to 6 days postinfection (dpi), p24 further declined in all groups treated with liposomal formulations compared to the HIV-infected control. Notably, both the HIV+liposome and HIV+liposomal CBD groups showed a significant reduction in p24 levels compared to the HIV+CBD group. However, no significant difference was observed between the HIV+liposome and HIV+liposomal CBD groups (Figure 9c). Moreover, LTR gene expression at 6 dpi (Figure 9d) did not show a statistically significant reduction compared to the infected group, suggesting a time-dependent plateau in transcriptional suppression. Overall, these results support that the liposomal nano formulation enhances the antiviral effectiveness of CBD against HIV infection, particularly at early stages (3 dpi).

Infected microglial cultures treated with the liposomal NF containing CBD showed a significant upregulation of secreted APOE protein at 3 days postinfection (dpi) compared to both untreated and HIV+CBD-treated cultures (Figure 10a), suggesting that the nano formulation enhances APOE expression as early as 3 dpi. In contrast, APOE levels were not significantly increased in cultures treated with free CBD alone. Furthermore, APOE secretion in the HIV-infected and HIV+CBD groups was significantly lower than in the HIV+liposomal CBD group at 3 dpi (Figure 10a).

Figure 10.

Effects of CBD-Liposomal NF on APOE relative expression. Results show the relative expression of the APOE protein secretion. Supernatants from cell treated with Liposomal nanoformulation to deliver 1 μM of CBD and free CBD in infected or noninfected C20 cells were collected after 3 (A) & 6 (B) days. We assessed the concentration of extracellular APOE protein using Speedy Human APOE ELISA Kit (Catalog # SE50006; Proteintech). All the data was analyzed using GraphPad Prism software 10. Outliers were identified by Rout method and cleaned data was used for analysis. The normality distribution was evaluated by Kolmogorov–Smirnov test, while statistical significance was calculated by ANOVA and Tukey’ s Multiple Comparison Test as a post hoc test. Differences were considered significant at p ≤ 0.05. Statistical significance is indicated as * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001; **** = P ≤ 0.0001. Only significance comparison are been showed.

However, this effect did not persist over time. At 6 dpi, the liposomal formulation failed to maintain elevated APOE secretion (Figure 10b). In contrast, cultures treated with free CBD showed significantly higher APOE levels compared to the control and HIV liposome groups. These results suggest that the stimulatory effect of the liposomal CBD formulation on APOE expression is transient, with peak activity observed at 3 dpi.

Given these findings and the well-established functional implications of APOE gene polymorphisms, we proceeded to genotype the APOE variants present in the C20 cell line. To this end, we designed human-specific primers targeting the three major APOE polymorphisms. Sequencing analysis confirmed that the C20 cells are homozygous for the APOE3 allele (Figure 11).

Figure 11.

APOE genotyping. Represents the results for sanger sequencing results from DNA after amplified the APOE gene. Our cells resulted homozygous APOE3. Cells also tested negative for mycoplasma. Created with Biorender (www.Biorender.com).

Considering that the nano formulation exhibits fluorescence within the same spectrum as DCFDA and given previous evidence of CBD’s brain-specific effects on mitochondrial respiration, we assessed reactive oxygen species (MitoROS) production across all treatment groups. HIV-infected cultures exhibited significantly higher MitoROS levels compared to the vehicle control, CBD liposome, liposome loaded with CBD, HIV+CBD, and HIV+liposomal CBD groups. No other significant differences in ROS production were observed among the remaining groups at this point (Figure 12a). At 6 dpi, treatment with CBD, liposome, and HIV+liposomal CBD resulted in a significant reduction in MitoROS levels compared to the HIV-infected group. Notably, the HIV+liposomal CBD group demonstrated a significantly greater reduction in oxidative stress relative to the HIV-only group, indicating a sustained antioxidant effect of the nanoformulated CBD (Figure 12b).

Figure 12.

Effects of CBD-Liposomal NF on mitochondrial ROS. C20 cells were treated with 1 μM of CBD, free and contained in liposome in presence and absence of HIV infection. After 3 (A) and 6 (B) days post infection, the MitoROS 580 assay (catalog #16052; Aat Bioquest) was performed following provider instructions. Cells were stained with Hoechst 33342 to normalize the MitoROS production per number of cells. Cells treated with H2O2 were used as positive control. All the comparison against H2O2 showed significant p-value. MitoROS 580 was measured at with Ex/Em = 510/580 using a Biotek Synergy HT multimode microplate reader instrument. All the data was analyzed using GraphPad Prism software 10. Outliers were identified and cleaned data was used for analysis. The normality distribution was evaluated by Kolmogorov–Smirnov test, while statistical significance was calculated by ANOVA and Dunns Multiple Comparison Test as a post hoc test. Differences were considered significant at p ≤ 0.05. Statistical significance is indicated as * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001; **** = P ≤ 0.0001.

Overall, the observed reduction in levels of p24 and LTR-HIV gene expression, along with increased APOE3 secretion, decreased mitochondrial ROS production, and efficient cellular uptake of the nano formulation, support the conclusion that the liposomal nano formulation significantly enhanced the therapeutic effectiveness of CBD, particularly at 3 dpi.

DISCUSSION

CBD is of interest due to its reported therapeutic effects, including pain relief, anti-inflammatory, antioxidant, anxiolytic, and antiepileptic properties, importantly, without psychotropic or addictive effects.^7,11 However, its potential in treating HIV infection and HAND remains unclear. To explore this, we examined CBD’s impact on oxidative stress in HIV-infected microglial cells using an Oxidative Stress Pathway Plus RT-PCR array. Based on the results, we selected a gene whose HIV-induced dysregulation was reversed by CBD for further investigation and developed an NF to assess whether it could enhance CBD’s efficacy, while also evaluating key hallmarks of HIV infection.

Our analysis revealed significant dysregulation of the APOE and TRAPPC6A genes in cultures treated with CBD compared to untreated HIV-infected controls. To the best of our knowledge, no prior study has reported experimental upregulation of APOE expression by CBD in the context of HIV. In silico studies suggest CBD may bind Alzheimer - related targets, including APOE, amyloid precursor protein, and presenilin-1.³⁷ APOE has antioxidant properties and exists in three main isoforms (APOE2, APOE3, and APOE4) differing in lipidation, which influences cholesterol transport, inflammation, and amyloid-β clearance.^38–48 APOE4, a major risk factor for late-onset Alzheimer’s, is linked to poor lipidation, higher amyloid-β binding, oxidative stress, inflammation, cholesterol, and cardiovascular risk.^46,48–50 Whereas, APOE2 promotes amyloid-β clearance and less oxidative damage,⁴¹ and gene therapy using APOE2 is under study to counter APOE4 effects.⁴² APOE3, found in 60–70% of people, is neutral in Alzheimer’s risk and linked to moderate cholesterol.⁴²

APOE affects susceptibility to various pathogens, including HSV, EBV, VZV, hepatitis C/E, malaria, Listeria monocytogenes, and Klebsiella pneumoniae.^51–54 Its role in HIV and HAND is mixed, likely due to APOE isoform variation, age, viral load, ART, and comorbidities.^55,56 APOE acts as an early restriction factor that inhibits HIV in macrophages, independently of some viral proteins and interferon genes.⁵¹ Silenced APOE increases HIV infectivity. All three isoforms reduce R5 HIV-1 envelope expression, with minimal effect on p24.⁵¹ The lipid-binding C-terminal region is essential for this inhibition,⁵¹ and SNPs lie outside this region.^51,55 Previous studies have shown, Tat uptake and LTR activation depend on APOE isoforms: APOE2/3 strongly reduce both, while APOE4 requires higher levels.⁵⁷ These effects are enhanced by HDL, suggesting liposomes with phospholipids and cholesterol may mimic HDL and boost the expression of APOE in the brain.⁵⁷

Conversely, APOE4 enhances CCR5-tropic HIV entry and is incorporated into virions from infected macrophages.³⁸ Unlike APOE3, lipidated APOE4 does not counteract Tat-induced neurotoxicity.⁵⁸ Clinically, APOE4 homozygosity is linked to faster HIV progression, while APOE3 is tied to slower progression.³⁸ Lack of APOE3 in HIV-positive individuals is more strongly associated with death than AIDS.^38,40 In our study, both free and liposomal CBD may exert anti-HIV effects partly by increasing APOE3 secretion.

CBD regulates cholesterol homeostasis by activating PPARγ, promoting cholesterol efflux, reducing influx, inhibiting foam cell formation, and lowering pro-inflammatory cytokines.^23,59,60 APOE aids in intracellular cholesterol transport. In aged astrocytes, CBD partially reverses lipid buildup, even with APOE4.^61–63 However, Kordi et al. (2024) found reduced APOE levels in amyloid-β–injected rats treated with CBD and high-intensity training.⁶⁴ CBD’s anti-inflammatory, antioxidant, and antiviral properties may indirectly support lipid balance and APOE expression.^15,60,65 APOE deficiency increases oxidative stress; for example, folate or iron imbalance worsens damage in APOE-knockout mice,^48,65 and synaptosomes from these mice are more vulnerable to amyloid-β–induced stress.⁴³

We recently reported that CBD significantly reduces LTR HIV gene expression in infected microglia.⁷ The HIV LTR region controls viral transcription, genome integration, and latency reactivation, as it contains enhancers and promoters.^66,67 Similarly, at 6 μM of CBD reduced HIV spread was reduced in long-term infected macrophages⁶⁸ and De Marino et al.⁹ also showed CBD decreased extracellular vesicle release from HIV-infected cells, suppressed viral transcription, and oxidative stress.⁹ Our results demonstrate that the CBD NF enhanced APOE production while reducing HIV LTR expression and p24 levels more rapidly than free CBD. These data suggest that the anti-HIV effects of CBD, both free and liposome-encapsulated, may be partially mediated by the early induction and secretion of APOE during the initial stages of infection.

Our liposomal formulation incorporates L-α-phosphatidylcholine (PC) from egg, known to enhance absorption and bioavailability.^69–71 PC and its metabolite choline, a precursor to acetylcholine, have demonstrated cognitive benefits, with supplementation improving performance in recent studies.⁷² Reduced PC levels in Alzheimer’s disease are associated with impaired synaptic function and elevated Aβ production.⁷³ The formulation also includes oleic acid (32%) and linoleic acid (18.2%), which promote membrane fluidity and may improve BBB permeability.^74–77 Cholesterol, included at 0.2 mg/300 μL, stabilizes membranes and reduces oxidative stress,^35,78 while our 2:1 lipid-to-cholesterol ratio supports nanoparticle stability³⁶ and drug release.^36,78 This composition produced nanoparticles ranging from 109–370 nm with high encapsulation efficiency and near-neutral surface charge, which may facilitate immune evasion and improved biocompatibility.²⁵ Liposomes exhibited an average particle size below 370 nm and a narrow size distribution. The polydispersity index (PDI) values were 0.200 for the blank liposomal nanoformulation and 0.187 for the CBD-loaded liposomal formulation, indicating a homogeneous population of vesicles. In dynamic light scattering measurements, the PDI ranges from 0.0 (perfectly monodisperse) to 1.0 (highly polydisperse). According to previous reports, PDI values ≤ 0.3 are generally accepted as indicative of a relatively uniform nanoparticle population, whereas values ≤ 0.2 are characteristic of a very narrow size distribution.^79,80 LC-MS analysis confirmed the presence and purity of CBD, with a consistent retention time and a prominent molecular ion peak at m/z 315.2348, along with characteristic fragments. The sharp, singular peak and absence of interference indicate minimal degradation and a stable formulation, confirming the suitability of LC-MS for CBD identification and quantification in future pharmacokinetic studies.⁷⁹ The lower size of CBD liposome formulation can be attributed to various factors which include the method of synthesis (lipid film formation followed by extrusion), concentration of CBD and phospholipids used for liposome synthesis. Additionally, the lipophilic and molecular structure of CBD may have resulted in a stable liposome. The lipid film containing CBD may have resulted in more compact vesicles and the interaction with the fatty acids tails of Phospholipids and have influenced the overall size of the formulation.⁷⁹

Furthermore, HIV infection alters membrane properties, including lipid organization and fluidity, which may reduce liposome uptake.^81–83 Nonetheless, we observed up to 40% uptake in HIV-infected cells, sufficient to increase APOE secretion and decrease p24 and LTR expression. At 6 dpi, the uptake peaked, coinciding with maximal suppression of HIV markers.

Oxidative stress is another key finding in this study. To our knowledge, no prior studies have explored the combined effects of CBD, APOE, liposomal NFs, and HIV infection. At 3 dpi, HIV markedly increased oxidative stress, which was significantly reduced by both free and liposomal CBD. This reduction paralleled lower levels of p24 and LTR expression, along with increased APOE3 expression.

The observed capacity of liposomal CBD nanoparticles to reduce oxidative stress and modulate HIV infection holds significant promise for advancing human health interventions. In individuals living with HIV (PLWH), even under effective antiretroviral therapy, persistent oxidative damage contributes substantially to non-AIDS comorbidities, including neurocognitive impairments and accelerated aging. Liposomal CBD formulations enhance the bioavailability and targeted delivery of cannabidiol, enabling more effective penetration of key reservoirs such as microglia and potentially the brain. This targeted approach may mitigate the production of reactive oxygen species, stabilize antioxidant defenses, and attenuate HIV-associated neuroinflammation. Moreover, liposomal encapsulation may improve the stability and half-life of CBD, allowing for lower doses to achieve therapeutic effects while minimizing systemic side effects and potential drug-drug interactions. Consequently, liposomal CBD could serve as a valuable adjunctive therapy, particularly for patients who continue to experience complications despite optimal viral control. Acknowledging these findings may provide valuable insights into translational strategies, particularly regarding the role of lipid-based delivery systems in enhancing the brain bioavailability of CBD through various routes of administration.

Finally, TRAPPC6A (Trafficking Protein Particle Complex Subunit 6A), one of the dysregulated genes, is part of the TRAPP complex,⁸⁴ which regulates protein trafficking between the endoplasmic reticulum and the Golgi apparatus.⁸⁵ Mutations in TRAPPC6A are associated with intracellular protein accumulation, impaired nonverbal reasoning, and neurodegenerative disorders such as Alzheimer’s disease.^84–86 Further research is needed to determine whether CBD modulates TRAPPC6A or related pathways in HIV-associated neurocognitive disorders.⁸⁷

Acknowledging these findings may provide valuable insights into translational strategies, particularly regarding the role of lipid-based delivery systems in enhancing the brain bioavailability of CBD through various routes of administration.

CONCLUSION

The current study demonstrates that cannabidiol reduces HIV infectivity in microglial cells. Furthermore, CBD induces upregulation of the APOE3 and TRAPPC6 genes, along with increased APOE3 protein secretion, specifically in HIV-infected microglia homozygous for the APOE3 genotype. Notably, liposomal CBD nano formulation further enhanced the anti-HIV effects of CBD, significantly increased APOE expression, and reduced intracellular reactive oxygen species levels. However, it remains unclear whether APOE3 directly contributes to the observed antiviral effects or whether its upregulation is part of a broader, multifactorial response involving both CBD and components of NF. To our knowledge, this is the first study to report a direct modulation of APOE expression by CBD in HIV-infected cells. These findings provide a promising foundation for future therapeutic strategies aimed at reducing HIV-associated infection and oxidative stress in the brain. Moreover, they highlight the potential for personalized treatment approaches, particularly in individuals homozygous for the APOE3 allele.

Supplementary Material

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsbiomaterials.5c01218.

The Supporting Information provides the functional group classification of genes in the RT² Profiler PCR Array Human Oxidative Stress Pathway Plus, along with fold-change expression data for genes differentially expressed among groups, including comparisons of each group to the control condition (PDF)

ABBREVIATIONS

HSV

herpes simplex

EBV

Epstein–Barr virus

VZV

varicella zoster virus

Data Availability Statement

The data sets generated for this study are available on request to the corresponding author.