A Potential Effect of Circadian Rhythm in the Delivery/Therapeutic Performance of Paclitaxel–Dendrimer Nanosystems

Citation: Albuquerque, T.; Neves, A.R.; Paul, M.; Biswas, S.; Vuelta, E.; García-Tuñón, I.; Sánchez-Martin, M.; Quintela, T.; Costa, D. A Potential Effect of Circadian Rhythm in the Delivery/Therapeutic Performance of Paclitaxel–Dendrimer Nanosystems. J. Funct. Biomater. 2023, 14, 362. https://doi.org/10.3390/ jfb14070362 Academic Editor: Syed Nasir Abbas Bukhari Received: 13 June 2023 Revised: 5 July 2023 Accepted: 9 July 2023 Published: 11 July 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Journal of Functional Biomaterials Article A Potential Effect of Circadian Rhythm in the Delivery/Therapeutic Performance of Paclitaxel–Dendrimer Nanosystems Tânia Albuquerque 1,†, Ana Raquel Neves 1,†, Milan Paul 2, Swati Biswas 2, Elena Vuelta 3,4,5 , Ignacio García-Tuñón4, Manuel Sánchez-Martin 3,5,6 , Telma Quintela 1,7 and Diana Costa 1,* 1CICS-UBI—Health Sciences Research Centre, Universidade da Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal; [email protected] (T.A.); [email protected] (A.R.N.); [email protected] (T.Q.) 2Department of Pharmacy, Nanomedicine Research Laboratory, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, Jawahar Nagar, Medchal, Hyderabad 500078, Telangana, India; [email protected] (M.P.); [email protected] (S.B.) 3Servicio de Transgénesis, Nucleus, Universidad de Salamanca, 37008 Salamanca, Spain; [email protected] (E.V.); [email protected] (M.S.-M.) 4IBSAL, Instituto de Investigación Biomédica de Salamanca, 37007 Salamanca, Spain; [email protected] 5Departamento de Medicina, Universidad de Salamanca, 37008 Salamanca, Spain 6Unidad de Diagnóstico Molecular y Celular del Cáncer, Instituto Biología Molecular y Celular del Cáncer (USAL/CSIC), 37007 Salamanca, Spain 7UDI-IPG-Unidade de Investigação para o Desenvolvimento do Interior, Instituto Politécnico da Guarda, 6300-559 Guarda, Portugal *Correspondence: [email protected] †These authors contributed equally to this work. Abstract: The circadian clock controls behavior and physiology. Presently, there is clear evidence of a connection between this timing system and cancer development/progression. Moreover, circadian rhythm consideration in the therapeutic action of anticancer drugs can enhance the effectiveness of cancer therapy. Nanosized drug delivery systems (DDS) have been demonstrated to be suitable engineered platforms for drug targeted/sustained release. The investigation of the chronobiologynanotechnology relationship, i.e., timing DDS performance according to a patient’s circadian rhythm, may greatly improve cancer clinical outcomes. In the present work, we synthesized nanosystems based on an octa-arginine (R8)-modified poly(amidoamine) dendrimer conjugated with the anticancer drug paclitaxel (PTX), G4-PTX-R8, and its physicochemical properties were revealed to be appropriate for in vitro delivery. The influence of the circadian rhythm on its cellular internalization efficiency and potential therapeutic effect on human cervical cancer cells (HeLa) was studied. Cell-internalized PTX and caspase activity, as a measure of induced apoptosis, were monitored for six time points. Higher levels of PTX and caspase-3/9 were detected at T8, suggesting that the internalization of G4-PTX-R8 into HeLa cells and apoptosis are time-specific/-regulated phenomena. For a deeper understanding, the clock protein Bmal1—the main regulator of rhythmic activity, was silenced by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. Bmal1 silencing was revealed to have an impact on both PTX release and caspase activity, evidencing a potential role for circadian rhythm on drug delivery/therapeutic effect mediated by G4-PTX-R8. Keywords: apoptosis; Bmal1 silencing; cancer therapy; caspases; circadian rhythm; nano-delivery systems; PAMAM 1. Introduction Although the understanding of cancer has increased over the past decades, it continues to be a leading cause of death worldwide and one of the major health scourges of the 21st century [ 1 ]. For decades, the main goal of oncology research has been the development J. Funct. Biomater. 2023,14, 362. https://doi.org/10.3390/jfb14070362 https://www.mdpi.com/journal/jfb J. Funct. Biomater. 2023,14, 362 2 of 20 of efficient therapies to eliminate cancer cells by the induction of programmed cell death. The apoptosis process can occur through two different pathways (intrinsic and extrinsic) both leading to the activation of caspases, proteins responsible to initiate and control this mechanism [2]. More recently, nanotechnology has been intensely explored for cancer treatment. Unlike conventional chemotherapeutics, nanoscale delivery systems have shown many advantages, such as improved stability and biocompatibility, precise targeting of tumor cells, increased cellular uptake, reduction in side effects, and drug resistance [ 3 , 4 ]. Therefore, research in the nanotechnology field offers safer and more efficient possibilities for the constant improvement of cancer therapy [ 5 ]. Several nanocarriers with distinct physiochemical properties and compositions have been designed for antitumor drug release. Some of those include polymer-based vectors, liposomes, micelles derived from polymers, carbon nanotubes, solid lipid nanoparticles, and magnetic nanoparticles [ 6 ]. To be significantly effective, they should be able to overpass biological barriers and target tumor tissues without losing substantial activity upon blood circulation [ 7 ]. Later, after cellular uptake, they should ideally be capable of endosomal escape and specific organelle targeting of the therapeutic agents [8]. Dendrimers are synthetic polymeric nanomaterials applied in both gene and drug delivery [ 9 ]. They are recognized as versatile carriers due to their unique structural features: a spherical polymeric core surrounded by multiple well-ordered geometrical branching molecules, where drugs can be encapsulated, and a periphery that can be functionalized with different ligands to enhance their stability, targeting ability, and tissue penetration [ 10 ]. These three-dimensional tree-like structures can be classified into “generations” (G) concerning the number of the branching in the dendrimer [ 11 ]. With an increase in generations, the molecular weight and number of reactive surface groups increase as well, creating dendrimers with different properties and possibilities of applications. Poly (amido amine) dendrimers (PAMAM), in particular, have been one of the most commonly used and have shown promising results for the delivery of poorly soluble anticancer drugs such as PTX [ 12 ], methotrexate (MTX) [ 13 ], doxorubicin [ 14 ], and docetaxel [ 15 ]. To further improve the cellular internalization of such constructs, Cell-Penetrating Peptides (CPPs) can be added. CPPs are small peptides originating from different sources that possess remarkable properties of cell penetration, facilitating the translocation of therapeutic agents into cells [ 16 , 17 ]. Cationic peptides are a class of CPPs that easily interact with the phospholipids, negative in charge, in the cell membrane, facilitating membrane translocation. For instance, octa-arginine (R8), a short-chain peptide with eight arginine residues was found to mediate intracellular uptake of the nano-constructs leading to the intracellular release of a high amount of PTX promoting its anticancer efficacy [ 12 , 18 ]. Chronotherapy has also gained much attention in the cancer research field as an emergent strategy to enhance current cancer treatments. It is based on the principle that the safety and efficacy of chemotherapeutics can be dependent on the time of their administration [ 19 ]. So far, several clinical trials have applied chronotherapeutic protocols for cancer treatment and have confirmed an optimal timing for therapy, improving its efficacy, diminishing drug toxicity, and enhancing patient survival [ 20 , 21 ]. In fact, it is well documented that the circadian rhythm exerts a role in cancer progression since its dysfunction was associated with the activation of intracellular inflammatory and oncogenic signaling pathways [ 22 , 23 ]. On the other side, circadian rhythms modulate many cellular and physiological processes including drug metabolism, absorption, transport, distribution, detoxification, and subsequent toxicity and efficacy of the treatment. The study of circadian fluctuations of the molecules that are involved in these processes allows for the estimation of a more suitable time for treatment administration. The coordination of circadian rhythms is controlled by biological clocks found in nearly every cell in the body. A molecular clock machinery consists of clock genes/proteins that integrate regulatory feedback loops in which they regulate their own expression. The main components of the clock include brain and muscle ARNT-like1 (BMAL1), circadian locomotor output cycles protein kaput (CLOCK), the proteins period J. Funct. Biomater. 2023,14, 362 3 of 20 (PER1, PER2, and PER3), and cryptochrome (CRY1 and CRY2). The transcription factor CLOCK:BMAL1 forms a dimer to promote the expression of PER and CRY, which, in turn, act as repressors of CLOCK:BMAL1 in the nucleus. The outcome is the circadian oscillation of many genes and proteins involved in several physiological processes, within a period of about 24 h [ 24 ]. A correlation between clock gene expression and the apoptotic effect of anticancer agents has been reported [ 25 ]. Slat et al. found that the anticancer drug temozolomide (TMZ) attained a maximum effect close to the daily peak of Bmal1 expression. Moreover, the deletion of the core clock gene abolished the rhythmicity in TMZ-induced apoptosis in vitro [ 25 ]. Another study using in vitro and in silico models linked irinotecan cytotoxicity, a drug for colorectal cancer, to clock gene Bmal1 expression, putting in evidence a role for circadian rhythms in cancer treatment [26]. An attractive approach to overcome the limitations of current chemotherapeutics would be the combination of chronobiology with nanotechnology; however, few works have addressed the influence of the circadian clock in the efficiency of nanoformulations, namely, the cell entry of the vector, its release pattern and therapeutic effect. In a previous work, the effect of the circadian clock on the cellular uptake of nanoparticles based on polyethylenimine (PEI) to deliver MTX and p53-plasmid DNA to HeLa and C33A cell lines was investigated [ 27 ]. The results revealed a specific time point, after cell synchronization, for the high performance of the drug/gene co-delivery system. In this work, we intended to continue the research on this topic and bring advances to the chronobiologynanotechnology relationship. Pursuing this challenge, we synthesized a PAMAM dendrimer of generation 4 (G4), in which PTX and R8 were coupled (G4-PTX-R8). After the physicochemical characterization of the dendrimer nanosystem, it was tested whether the time point of delivery affected the amount of both cell-associated PTX and caspase activity in HeLa cells. Furthermore, we successfully performed Bmal1 silencing on HeLa cells to clarify the impact of the circadian clock on cellular uptake and apoptosis. Our data demonstrated significant differences, in the profile of these phenomena, between wild-type and knockout cells, suggesting a potential role for the circadian clock in the performance of a G4-PTX-R8 delivery system. 2. Materials and Methods 2.1. Materials Dulbecco ´ s Modified Eagle ´ s Medium with Ham’s F-12 Nutrient Mixture ( DMEM-F12 ) with L-glutamine was purchased from CORNING (New York, NY, USA). Cervical cancer HeLa cells were obtained from ATCC (Manassas, VA, USA). 3-(4,5dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) was supplied by Sigma-Aldrich (St. Louis, MO, USA). PTX was purchased by MedChemExpress (Monmouth Junction, NJ, USA). For G4-PTX-R8 synthesis, the dendrimer of fourth generation containing ethylenediamine core groups and surfaced amino groups (G4 PAMAM) was acquired from Dendritech (Midland, MI, USA). N-hydroxysuccinimide and N-(3-Dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride (EDC. HCl, 98%) were supplied by Sigma Aldrich Chemicals (St. Louis, MO, USA). Finally, the cellulose dialysis membrane (MWCO 3.5 KDa) was obtained from Spectrum Laboratories, Inc. (Dominguez, CA, USA). All other commercially purchased solvents and chemicals were of analytical grade. The reagents required for CRISPR-Cas9 technology (single guide RNAs (crRNAs), ATTO-labelled tracrRNA, and Cas9) were obtained from IDT (IA, Coralville, IA, USA); Buffer R and the primers for Bmal1 were purchased from Invitrogen (Carlsbad, CA, USA). The ApoAlertTM Caspase-3 Colorimetric assay kit was purchased from Clontech Lab (Orange, CA, USA), and Caspase-Glow®9 Assay from Promega (Madison, WI, USA). 2.2. Preparation of PTX-2-Hemisuccinate (PTX-SA) The steps for the formation of the G4 dendrimer conjugate are illustrated in Figure 1. The PTX-2 ´ -hemisuccinate was synthesized by activating the 2 0 -OH group of PTX using succinic anhydride (SA). Briefly, to 25 mg of PTX in Dichloromethane (DCM, 2 mL), 4.4 mg J. Funct. Biomater. 2023,14, 362 4 of 20 of SA was added, in the presence of dry pyridine, at a mol ratio of 1:1.5. This reaction was continuously mixed for 3 days [ 28 ]. Next, the PTX-SA was extracted from the aqueous reaction mixture using ethyl acetate, and the solvent was evaporated under vacuum, resulting in a white powder with an ~85% yield. The product was identified by thinlayer chromatography using silica-coated plate and solvent system. Dichloromethane: methanol. 85:15. J. Funct. Biomater. 2023, 14, x FOR PEER REVIEW 5 of 21 Figure 1. Illustration representing the main three steps in the preparation of the multifunctional G4PTX-R8 complex. 2.5. Nuclear Magnetic Resonance (NMR) Spectroscopy of PAMAM G4-PTX-R8 Complexes NMR was employed to characterize the developed conjugates, using an NMR Spectrometer (400 MHz, Bruker, Billerica, MA, USA). The samples of G4, PTX-SA, and G4PTX-R8 (10 mg/mL) were dissolved in the solvent CDCl3 and the spectra were obtained after 128 scans [30]. 2.6. Circular Dichroism (CD) Spectroscopy The secondary structure of R8 and G4-PTX-R8 was analyzed, under a nitrogen atmosphere using a CD spectrometer (Shimadzu, Japan) with a scanning range of 200–260 nm. The spectrum was compared with the standard R8 to confirm the presence of R8 in the formulation [31]. 2.7. Zeta Potential and Particle Size The average zeta potential and particle size of the G4-PTX-R8 were determined by the principle of dynamic light scattering using Zetasizer 3600 (Malvern Instruments Ltd., Malvern, UK). The zeta potential, particle size, and polydispersity index (PdI) were measured before and after lyophilizing the sample. The sample was diluted in Milli-Q water before analysis [32]. 2.8. Morphological Analysis Using SEM For the study of the G4-PTX-R8 surface morphology, Scanning Electron Microscopy (SEM, NOVA NANOSEM 450) analysis was performed. For sample preparation, a thin layer was evenly distributed and fixed to an aluminum stub with an adhesive carbon tape. Then, the stub was coated with the desired gold thickness and analyzed with a 20 kV accelerating voltage [33]. 2.9. Cell Culture For the in vitro experiments, cervical cancer HeLa cells were grown in 25 cm 3 T-flasks as described before [27]. HeLa cells (including knockout HeLa cells) were grown with DMEM-F12 supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% Figure 1. Illustration representing the main three steps in the preparation of the multifunctional G4-PTX-R8 complex. 2.3. Preparation of PAMAM G4-PTX The cross-linking agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/ N-hydroxysuccinimide (EDC/NHS) was used to activate the PTX-2-hemisuccinate in 2 mL of Dimethylformamide (DMF). The reaction occurred for 6 h at room temperature [29]. Following that, 50 mg of G4 PAMAM Dendrimer in DMF was transferred to the activated PTX-NHS ester, dropwise under nitrogen. The PAMAM G4: PTX-SA mol ratio was 1:4. The reaction continued overnight. Next, using a vacuum, the DMF was vaporized. Then, the solution was added to a dialysis membrane (3500 Da MWCO) and dialyzed for 48 h. Afterward, the solution was lyophilized, and a white powder was obtained. A yield of ~75% was obtained. 2.4. Preparation of PAMAM G4-PTX-R8 Into the solution of R8 (9.87 mg, 0.0078 mmol) and trimethylamine (20 µ L) in DMF, EDC/NHS (3 mol equivalent of the R8) G4-PTX (40 mg, 0.0026 mmol) was combined following the activation step. The reaction was continued overnight, followed by evaporation of DMF. Finally, using a cellulose ester membrane (MWCO. 3.5 KDa), the crude product was dialyzed against water, and the final mixture was lyophilized. A pure product was obtained with a yield of ~82%. An illustration with the main steps of the preparation of the dendrimer complex is represented in Figure 1. J. Funct. Biomater. 2023,14, 362 5 of 20 2.5. Nuclear Magnetic Resonance (NMR) Spectroscopy of PAMAM G4-PTX-R8 Complexes NMR was employed to characterize the developed conjugates, using an NMR Spectrometer (400 MHz, Bruker, Billerica, MA, USA). The samples of G4, PTX-SA, and G4-PTXR8 (10 mg/mL) were dissolved in the solvent CDCl3 and the spectra were obtained after 128 scans [30]. 2.6. Circular Dichroism (CD) Spectroscopy The secondary structure of R8 and G4-PTX-R8 was analyzed, under a nitrogen atmosphere using a CD spectrometer (Shimadzu, Japan) with a scanning range of 200–260 nm. The spectrum was compared with the standard R8 to confirm the presence of R8 in the formulation [31]. 2.7. Zeta Potential and Particle Size The average zeta potential and particle size of the G4-PTX-R8 were determined by the principle of dynamic light scattering using Zetasizer 3600 (Malvern Instruments Ltd., Malvern, UK). The zeta potential, particle size, and polydispersity index (PdI) were measured before and after lyophilizing the sample. The sample was diluted in Milli-Q water before analysis [32]. 2.8. Morphological Analysis Using SEM For the study of the G4-PTX-R8 surface morphology, Scanning Electron Microscopy (SEM, NOVA NANOSEM 450) analysis was performed. For sample preparation, a thin layer was evenly distributed and fixed to an aluminum stub with an adhesive carbon tape. Then, the stub was coated with the desired gold thickness and analyzed with a 20 kV accelerating voltage [33]. 2.9. Cell Culture For the in vitro experiments, cervical cancer HeLa cells were grown in 25 cm 3 T-flasks as described before [ 27 ]. HeLa cells (including knockout HeLa cells) were grown with DMEMF12 supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% (V/V) of a penicillin/streptomycin solution (100 µ g/mL). Cells were incubated at 37 ◦C , under a 5% CO 2 humidified atmosphere, to promote cellular growth until reaching confluency. 2.10. Cytotoxicity Study G4-PTX-R8 dendrimer powder was dissolved in DMSO:water 0.8:0.2 for posterior studies. To evaluate the cytotoxicity of free PTX and the G4-PTX-R8 dendrimer on HeLa, an MTT assay was performed. HeLa cells were seeded at a density of 5 × 10 3 cells/well in 96-well plates, and the day before the experiments, the culture medium was changed to DMEM-F12 supplemented with 10% FBS without antibiotics to enhance the cellular uptake [34,35]. Different formulations of PTX and G4-PTX-R8 ranging from 0 to 100 ug/mL were prepared and added to the cells during 6 h. Afterward, medium was replaced, and cells were allowed to grow at 37 ◦ C and 5% CO 2 for 48 h. After that period, MTT was added to each well at a final concentration of 0.5 mg/mL and incubated for a further 4 h. Furthermore, the culture medium was discarded and dimethyl sulfoxide (DMSO, 100 µ L) was added to dissolve the formazan crystals. Thereafter, the plates were shaken and a purple color was produced. The absorbance was then read at 570 nm using a Benchmark Microplate Reader (BioRad, Vienna, Austria). As a negative control of the experiment, wells with no treated cells were considered and cells treated only with DMSO were used as positive control. The medium without cells was set up as zero absorbance and used for spectrophotometer calibration. The cell viability (%) was therefore calculated in relation to the control wells by the formula: [A]test/[A]control × 100, [A]test being the absorbance of the test sample and [A]control the absorbance of the positive control sample. J. Funct. Biomater. 2023,14, 362 6 of 20 Moreover, the half maximal inhibitory concentration (IC 50 ) was assessed for free PTX and the G4-PTX-R8 complex. 2.11. In Vitro Studies to Acess Circadian Rhythms Impact on Cellular Uptake of the Nanosystem For in vitro studies, HeLa cells were seeded at a density of 5 × 10 4 cells/3.8 cm 2 in 12well microplates and grown until reaching 70–90% of confluence. Before the experiments, cells were synchronized for 2 h with 0.1 µ M of dexamethasone. Then, the medium was removed and cells returned to normal conditions [ 27 ]. Cells were incubated with free PTX or the G4-PTX-R8 complex at the IC50 concentration, calculated for G4-PTX-R8. The delivery was performed at T0 (immediately after cell synchronization) and every 4 h during 24 h (T4; T8; T12; T16; and T20). After 6 h of incubation, cells were either collected at the different time points for the cell-associated PTX absorbance measurement or cells returned to usual culture condition and left to grow during 48 h for subsequent assays. 2.12. Cell-Associated PTX To evaluate the cellular uptake of PTX in HeLa cells at the different time points, cellassociated PTX was measured. After collecting the cells, the pellet was rinsed in phosphatebuffered saline (PBS) and collected. Then, cell lysis was performed by incubation with a 1% Triton X-100 solution for 30 min at 37 ◦ C. Lastly, the solution was pipetted into a black plate, and intracellular PTX was determined by reading the absorbance at 230 nm using a Shimadzu UV-Vis 1700 spectrophotometer (Biorad). Cells without treatment were used as control [34]. 2.13. Bmal1 Silencing on Hela Cell Line The gene editing technique CRISPR-Cas 9 was used with the purpose of studying the effect of Bmal1 deletion on the efficacy of the G4-PTX-R8 system at all the 6 time points. Two crRNAs (Integrated DNA Technology) were designed to generate a small deletion (140 bp) targeting exon 8 (Supplementary Material, Table S1). For the complex formation, equimolar amounts were mixed to a final duplex concentration of 44 µ M. Then, the mixture was heated to 95 ◦ C for 5 min and then the temperature was ramped down to 25 ◦ C on a thermocycler. Next, 18 pmol of Cas9 (Integrated DNA Technology) was added to the initial duplex reaction (22 pmol) to a final volume of 1 µ L for electroporation and the mixture was set at room temperature (RT) for 1 h. Before electroporation, 2 µ L of 10.8 µ M of Electroporation Enhancer and 9 µ L of cell suspension were added to the final reaction. An amount of 2 × 10 5 cells was electroporated in a Neon TM Transfection System (Invitrogen, Waltham, CA, USA) following the manufacturer’s instructions and using the electroporation parameters of 1005 V, 35 ms, and 2 pulses. The day after transfection, cells were observed under confocal microscopy to confirm the efficiency of electroporation. Subsequently, cells were sorted with a FACSaria ( BD Biosciences , San Jose, CA, USA). This step was performed to select the gene-targeted cells by separation of the Bmal1 cell population from the control with only Cas9. Results were analyzed using FlowJo software. After the sorting, cells were collected, and one single cell was seeded in a new plate and left to grow into colonies. The confirmation of the knockout harboring clones was first assessed by conventional PCR (primer sequence on Supplementary Material, Table S2). The AllPrep DNA Kit (Qiagen) was used for the extraction of Genomic DNA according to the manufacturer’s protocol. The PCR products were cleaned with the NZYGelpure kit (NZYTech, Lisbon, Portugal) and sequenced by the Sanger method [36]. 2.14. Caspase-3 and Caspase-9 Activity Assay Caspase-3 and caspase-9 activity were measured on wild-type and knockout HeLa cells following the provided instructions. In brief, after the incubation with free PTX or G4-PTX-R8, cells were allowed to grow for 24 h. An incubation with 1 µ M of staurosporine, under the same conditions, was used as a positive control. J. Funct. Biomater. 2023,14, 362 7 of 20 The caspase-3 activity was determined by using a caspase colorimetric assay Kit (ApoAlertTM), by measuring the absorbance at 405 nm of p-nitroaniline (p-NA) after cleavage from the substrate DEVD-pNA. A Luminescent Assay (Caspase-Glow ® 9) was applied for the determination of Caspase-9 activity following the provided protocol [34]. 2.15. Statistical Analysis For the comparison between the distinct experimental groups, one-way/two-way analysis of variance (ANOVA) and Bonferroni test were employed. The data were analyzed in GraphPad Prism v.8.01 software (San Diego, CA, USA). Statistically significant differences were considered for a p-value below 0.05 (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p≤0.0001). The rhythmicity in PTX intracellular uptake and caspase activity was analyzed with CircWave v1.4 analysis software (Dr. Roelof A. Hut, Groningen, The Netherlands) by a harmonic regression method, assuming a period of 24 h, and with alpha set at 0.05. Statistically significant rhythms were considered for a p-value below 0.05. 3. Results 3.1. Preparation and Characterization of Multifunctional Dendrimer Conjugate The multifunctional dendrimer conjugate was synthesized as described in Figure 1and characterized by NMR spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray Photoelectron spectroscopy (XPS), CD Spectroscopy, Gel Permeation Chromatography (GPC), Zeta potential and Particle size determination, and SEM (detailed description available in Supplementary Material). Figure 2b shows the 1H-NMR spectra of PTX-SA displaying a peak at 2.5–2.8 ( δ ), which indicates the coupling of succinic acid to the PTX. The spectra of G4 and G4-PTX-R8 present a signal at ppm 7–8.5 ( δ ), which corresponds to the aromatic groups of the PTX. In Figure 2c,d, the signals from the dendrimer protons are observed at ppm 1.5–3.5 ( δ ). Finally, the signals visible at ppm 1–1.5 ( δ ) relate to the methylene protons of R8 [ 12 ]. Furthermore, FTIR and XPS validated the conjugation of PTX and R8 to the dendrimer (Figures S1 and S2 and Table S3, all presented in Supplementary Material)). J. Funct. Biomater. 2023, 14, x FOR PEER REVIEW 8 of 21 Figure 2b shows the 1H-NMR spectra of PTX-SA displaying a peak at 2.5–2.8 (δ), which indicates the coupling of succinic acid to the PTX. The spectra of G4 and G4-PTXR8 present a signal at ppm 7–8.5 (δ), which corresponds to the aromatic groups of the PTX. In Figure 2c,d, the signals from the dendrimer protons are observed at ppm 1.5–3.5 (δ). Finally, the signals visible at ppm 1–1.5 (δ) relate to the methylene protons of R8 [12]. Furthermore, FTIR and XPS validated the conjugation of PTX and R8 to the dendrimer (Figures S1 and S2 and Table S3, all presented in SM). Figure 2. Thin-layer chromatography of PTX-SA conjugate (a), 1H-NMR spectra of PTX-SA (b), PAMAM G4 (c), and G4-PTX-R8 (d). To further confirm the presence of R8 in the formulation, CD Spectroscopy was performed. The alpha-helix and beta-sheet values obtained for standard R8 were 70.71 ± 0.79 and 10.21%, respectively. The R8-conjugated polymer, G4-PTX-R8, displayed the values as 59.32 ± 0.12 and 14.32%, respectively (Figure 3). The decrease in the CD signal in the G4-PTX-R8 conjugate indicated the binding of molecules to the peptide, which induced its conformational change. However, the secondary structure remains the same with the predominance of alpha-helical structure. Figure 2. Thin-layer chromatography of PTX-SA conjugate ( a ), 1H-NMR spectra of PTX-SA (b), PAMAM G4 (c), and G4-PTX-R8 (d). To further confirm the presence of R8 in the formulation, CD Spectroscopy was performed. The alpha-helix and beta-sheet values obtained for standard R8 were 70.71 ±0.79 J. Funct. Biomater. 2023,14, 362 8 of 20 and 10.21%, respectively. The R8-conjugated polymer, G4-PTX-R8, displayed the values as 59.32 ± 0.12 and 14.32%, respectively (Figure 3). The decrease in the CD signal in the G4-PTX-R8 conjugate indicated the binding of molecules to the peptide, which induced its conformational change. However, the secondary structure remains the same with the predominance of alpha-helical structure. J. Funct. Biomater. 2023, 14, x FOR PEER REVIEW 9 of 21 Figure 3. Representation of CD spectra of R8 and G4-PTX-R8. The presence of R8 in the final formulation was confirmed, as well as the structural integrity following conjugation. The relative molecular weights of the synthesized product were determined by using GPC. The average molecular weight of the dendrimer complex was 19313 Da (Table S4, SM). The GPC data analysis also indicated that 2.48 molecules of R8 were conjugated to one molecule of G4-PTX (Figure S3, SM) in the stated experimental conditions. Finally, the zeta potential and particle size of G4-PTX-R8 formulations are presented in Table 1. The morphological analysis using SEM (Figure 4) displayed uniform spherical morphology of the dendrimer, and the zeta potential revealed the positive surface charge of the conjugate. Table 1. Size, PdI, and zeta potential value obtained for PAMAM G4-PTX-R8 dendrimer. Zeta Potential (mV) PdI Size (nm) PAMAM G4-PTX-R8 +9.325 0.287 23.74 ± 0.454 Figure 4. SEM image of PAMAM G4-PTX-R8 dendrimer. 3.2. Cytotoxicity Studies To determine the efficacy of the PTX conjugate in inhibiting cancer cell viability, an MTT assay was performed in HeLa cells. Cells were treated with free PTX or G4-PTX-R8 at concentrations ranging from 0 to 100 µg/mL and incubated for 48 h, as presented in Figure 5. Cells that did not receive the treatment were taken as a positive control for Figure 3. Representation of CD spectra of R8 and G4-PTX-R8. The presence of R8 in the final formulation was confirmed, as well as the structural integrity following conjugation. The relative molecular weights of the synthesized product were determined by using GPC. The average molecular weight of the dendrimer complex was 19313 Da (Table S4, Supplementary Material). The GPC data analysis also indicated that 2.48 molecules of R8 were conjugated to one molecule of G4-PTX (Figure S3, Supplementary Material) in the stated experimental conditions. Finally, the zeta potential and particle size of G4-PTX-R8 formulations are presented in Table 1. The morphological analysis using SEM (Figure 4) displayed uniform spherical morphology of the dendrimer, and the zeta potential revealed the positive surface charge of the conjugate. Table 1. Size, PdI, and zeta potential value obtained for PAMAM G4-PTX-R8 dendrimer. Zeta Potential (mV) PdI Size (nm) PAMAM G4-PTX-R8 +9.325 0.287 23.74 ±0.454 3.2. Cytotoxicity Studies To determine the efficacy of the PTX conjugate in inhibiting cancer cell viability, an MTT assay was performed in HeLa cells. Cells were treated with free PTX or G4-PTX-R8 at concentrations ranging from 0 to 100 µ g/mL and incubated for 48 h, as presented in Figure 5. Cells that did not receive the treatment were taken as a positive control for cellular viability. The toxicity was accessed by calculating the IC 50 value from the different concentrations using a nonlinear curve fitting algorithm. The results of the assay demonstrated that there is evidence of a cellular viability decrease in time as the PTX concentration increases (Figure 5). J. Funct. Biomater. 2023,14, 362 9 of 20 J. Funct. Biomater. 2023, 14, x FOR PEER REVIEW 9 of 21 Figure 3. Representation of CD spectra of R8 and G4-PTX-R8. The presence of R8 in the final formulation was confirmed, as well as the structural integrity following conjugation. The relative molecular weights of the synthesized product were determined by using GPC. The average molecular weight of the dendrimer complex was 19313 Da (Table S4, SM). The GPC data analysis also indicated that 2.48 molecules of R8 were conjugated to one molecule of G4-PTX (Figure S3, SM) in the stated experimental conditions. Finally, the zeta potential and particle size of G4-PTX-R8 formulations are presented in Table 1. The morphological analysis using SEM (Figure 4) displayed uniform spherical morphology of the dendrimer, and the zeta potential revealed the positive surface charge of the conjugate. Table 1. Size, PdI, and zeta potential value obtained for PAMAM G4-PTX-R8 dendrimer. Zeta Potential (mV) PdI Size (nm) PAMAM G4-PTX-R8 +9.325 0.287 23.74 ± 0.454 Figure 4. SEM image of PAMAM G4-PTX-R8 dendrimer. 3.2. Cytotoxicity Studies To determine the efficacy of the PTX conjugate in inhibiting cancer cell viability, an MTT assay was performed in HeLa cells. Cells were treated with free PTX or G4-PTX-R8 at concentrations ranging from 0 to 100 µg/mL and incubated for 48 h, as presented in Figure 5. Cells that did not receive the treatment were taken as a positive control for Figure 4. SEM image of PAMAM G4-PTX-R8 dendrimer. J. Funct. Biomater. 2023, 14, x FOR PEER REVIEW 10 of 21 cellular viability. The toxicity was accessed by calculating the IC50 value from the different concentrations using a nonlinear curve fitting algorithm. The results of the assay demonstrated that there is evidence of a cellular viability decrease in time as the PTX concentration increases (Figure 5). Figure 5. Dose–response curves and cellular viability of HeLa cervical cancer cells after 48 h of treatment with free PTX (a) and the G4-PTX-R8 complex (b). Each data point represents the mean value calculated with the data obtained from independent measurements (n = 3, mean ± SD), which were analyzed by one-way ANOVA with the Bonferroni test (**** < 0.0001 relative to control). The determination of the median inhibitory concentration is used to estimate how effective a given anticancer drug is at reducing cellular viability. The value of IC50 is lower for G4-PTX-R8, meaning that a lower dose is required to produce the same toxicity as free PTX. The subsequent experiments were conducted based on the IC50 value achieved for G4-PTX-R8. 3.3. The Impact of Circadian Rhythm on Cellular Uptake/Internalization 3.3.1. Determination of Cell-Associated PTX Cell synchronization was confirmed after analyzing the expression of Bmal1 over 24 h (Figure S4). To evaluate the impact of the circadian rhythm on PTX cellular uptake/internalization, the released PTX in the cytosol of HeLa cells after incubation with the G4PTX-R8 dendrimer or free PTX was measured for each time point indicated above. Figure 6a shows the obtained results. From the presented results, regarding the cell-associated PTX absorbance measure, we can infer that both free drug and dendrimer-associated drug are internalized by HeLa, however, in a different extension and differently over time. Figure 5. Dose–response curves and cellular viability of HeLa cervical cancer cells after 48 h of treatment with free PTX ( a ) and the G4-PTX-R8 complex ( b ). Each data point represents the mean value calculated with the data obtained from independent measurements (n = 3, mean ± SD), which were analyzed by one-way ANOVA with the Bonferroni test (**** p< 0.0001 relative to control). J. Funct. Biomater. 2023,14, 362 16 of 20 Given our findings, we believe that a way to make cancer therapy more effective should include the optimization of nanosystem design/development to ensure drug delivery at the appropriate time of the day, in accordance with the patient´s circadian rhythm. Needless to say, the suitable time for in vivo administration should take several factors into account. Upon administration, nanoparticles will be exposed to pharmacokinetic phenomena, many of which are under circadian regulation. All these factors are supposed to impact drug toxicity and efficacy and must be considered for chronotherapy applications to enhance cancer therapy outcomes. 5. Conclusions The attractive concept of “Chronotherapy” has been explored to enhance cancer therapy outcomes. In parallel, to overcome the major obstacles of conventional therapies, a variety of nanosystems have been considered for the targeted drug release to cancer cells. In this work, we developed nanocomplexes based on a PAMAM dendrimer of generation 4 for PTX delivery into HeLa cancer cells. Regardless, these molecules can also face some challenges, such as membrane permeability. Consequently, the R8 peptide was successfully conjugated on the dendrimer surface. In our study, the efficiency of dendrimer cellular uptake was directly correlated with higher PTX absorbance found in the cytosol. We noted a superior PTX internalization when PTX was coupled with PAMAM and R8 rather than when used alone. Therefore, the dendrimer is a suitable vehicle for the release of poorly soluble drugs. To unveil the effect of the circadian rhythm on the efficiency of cellular internalization, we measured cell-associated PTX and caspase activity on wild-type HeLa and Bmal1silenced cells. The obtained results demonstrated higher delivery of PTX at T8 and T12 and superior caspase-3 activity at T8, and at T8 and T12 for caspase-9 activity. As for caspase activity, only caspase-3 expression was compromised by the Bmal1 knockdown in HeLa cells. Also, the knockdown maintained a rhythmic pattern for PTX internalization and caspase activity, however, to a much less extent. We concluded that Bmal1 appears to be necessary for the efficient uptake of the dendrimer and for the execution of programmed cell death. Bmal1 can either promote the cellular internalization of PTX, leading to the activation of apoptosis, or can increase susceptibility to the drug, leading to increased apoptosis at a specific time point. Although our results do not cover a full understanding of the role of Bmal1, our work strongly suggests the influence of Bmal1 on the performance of PTX/G4-PTX-G8 cellular uptake, PTX delivery, and, consequently, apoptosis induction, and, thus, therapeutic effect. The reported data should instigate the design of novel delivery systems displaying a more precise targeting to cancer cells, at a more favorable time. If translated and implemented in the clinic, it could have an exponential impact on cancer therapy. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jfb14070362/s1, Figure S1: FTIR spectra of G4, PTX-SA, G4PTX, and G4-PTX-R8; Table S1: crRNA sequences; Figure S2: XPS spectra of G4, G4-PTX, and G4-PTX-R8; Table S2: Primers designed for RT-PCR; Figure S3: GPC thermogram of G4, G4-PTX, and G4-PTX-R8; Table S3: XPS data of PAMAM G4-PTX-R8 dendrimer; Figure S4: CircWave analysis of bmal1 rhythmicity after HeLa cell synchronization; Table S4: GPC data of PAMAM G4-PTXR8 dendrimer; Figure S5: Genomic map of Bmal1 showing the sequence of the deleted fragment from exon 8 in grey and RT-PCR products of the isolated clones; Table S5: Statistic analyses of Bonferroni’s multiple comparisons test for PTX internalization on HeLa cells; Figure S6: Western blot analysis of BMAL1 protein expression in HeLa cells; Table S6: Results of CircWave analysis of cell-associated PTX absorbance for each time point; Figure S7: Circadian oscillations are statistically significant as analyzed with CircWave for PTX and the G4-PTX-R8 complex; Table S7: Statistic analyses of Bonferroni’s multiple comparisons test for PTX and G4-PTX-R8 internalization on HeLa knockout cells; Figure S8: Circadian oscillations are statistically significant as analyzed with CircWave for caspase-3 and caspase-9 activity after incubation with PTX and G4-PTX-R8; Table S8: Statistic J. Funct. Biomater. 2023,14, 362 17 of 20 analyses of Bonferroni’s multiple comparisons test for caspase activity on wild-type and knockout after incubation with free PTX or G4-PTX-R8. Author Contributions: Conceptualization, M.S.-M., T.Q. and D.C.; methodology, T.A., A.R.N., M.P., S.B., E.V., I.G.-T., M.S.-M., T.Q. and D.C.; software, T.A., A.R.N., M.P., S.B., E.V., I.G.-T., M.S.-M., T.Q. and D.C.; validation, S.B., E.V., I.G.-T., M.S.-M., T.Q. and D.C.; formal analysis, T.A., A.R.N., M.P., S.B., E.V., I.G.-T., M.S.-M., T.Q. and D.C.; investigation, T.A., A.R.N., M.S.-M., T.Q. and D.C.; resources, S.B., I.G.-T., M.S.-M., T.Q. and D.C.; data curation, T.A., A.R.N., M.P., S.B., E.V., I.G.-T., M.S.-M., T.Q. and D.C.; writing—original draft preparation, T.A., A.R.N. and D.C.; writing—review and editing, T.A., A.R.N., S.B., E.V., M.S.-M., T.Q. and D.C.; visualization, T.A., A.R.N., M.P., S.B., E.V., I.G.-T., M.S.-M., T.Q. and D.C.; supervision, S.B., M.S.-M., T.Q. and D.C.; project administration, S.B., M.S.-M., T.Q. and D.C.; funding acquisition, S.B., M.S.-M., T.Q. and D.C. All authors have read and agreed to the published version of the manuscript. Funding: D. Costa acknowledges the Fundação para a Ciência e a Tecnologia (FCT) her 2021.03946.CEECIND Researcher Contract. T. Albuquerque (SFRH/BD/148406/2019) and A. Neves (2020.08310.BD) acknowledge their FCT PhD grants, co-supported by the European Social Fund via Programa Operacional Regional Centro. Swati Biswas acknowledges the Indian Council of Medical Research (ICMR) for providing funding through a research grant (F-20218945). Milan Paul acknowledges ICMR for providing the senior research fellowship (45/11/2022/NAN/BMS). The presented research was developed within the scope of CICS-UBI projects UIDB/00709/2020 and UIDP/00709/2020, financed by national funds through the Portuguese FCT/MCTES. Data Availability Statement: All data is provided in the principal document of the manuscript or as supplementary materials. Conflicts of Interest: The authors declare no conflict of interest. References 1. Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. 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