Report of the 2023 AACP Council of Deans Taskforce on Pharmacy Research and Scholarship.

OBJECTIVE: The objective of this review is to provide the conclusions from the American Association of Colleges of Pharmacy (AACP) Council of Deans (COD) Taskforce on Research and Scholarship. FINDINGS: The charges and the findings of the committee are: (1) Define the scholarship needs/opportunities to strengthen the outputs. The committee recommends that AACP update its definitions of research/scholarship to include discovery, integration, application/practice, and teaching/learning. A deployed survey demonstrated a high Special Interest Groups research/scholarship interest. (2) Assemble a toolkit of grant and scholarship resources to assist colleges/schools. The AACP should update the existing funding opportunity listing and combine it with additional resources. (3) Create a framework for effective research collaboration and mentorship. The AACP should identify key areas of pharmacy research and experts to serve as mentors and to meet with external stakeholders. (4) and (5) Consider the need for and purpose of a COD standing committee for research and scholarship. Explore the value of a formal research dean's subcommittee. It was recommended that AACP form a research/scholarship committee or Special Interest Groups and create the Pharmacy Scholarship, Research, and Graduate Education pre-meeting to the Interim Meeting. (6) Identify key statements/outputs of the COD that need to be prepared for publication/sharing. We recommended the key statement/outputs in the areas of discovery, integration, application/practice, and teaching and learning. SUMMARY: The taskforce reviewed the state of research and scholarship across the Academy and provided recommendations with the goal of advancing research across all areas of the pharmacy profession.

Identification of Key Amino Acids that Impact Organic Solute Transporter α/ß (OSTα/ß).

Organic solute transporter α/ß (OSTα/ß) is a bidirectional bile acid transporter localized on the basolateral membrane of hepatic, intestinal, and renal epithelial cells. OSTα/ß plays a critical role in intestinal bile acid reabsorption and is upregulated in hepatic diseases characterized by elevated bile acids, whereas genetic variants in SLC51A/B have been associated with clinical cholestasis. OSTα/ß also transports and is inhibited by commonly used medications. However, there is currently no high-resolution structure of OSTα/ß, and structure-function data for OSTα, the proposed substrate-binding subunit, are lacking. The present study addressed this knowledge gap and identified amino acids in OSTα that are important for bile acid transport. This was accomplished using computational modeling and site-directed mutagenesis of the OSTα subunit to generate OSTα/ß mutant cell lines. Out of the 10 OSTα/ß mutants investigated, four (S228K, T229S, Q269E, Q269K) exhibited decreased [3H]-taurocholate (TCA) uptake (ratio of geometric means relative to OSTα/ß wild type (WT) of 0.76, 0.75, 0.79, and 0.13, respectively). Three OSTα/ß mutants (S228K, Q269K, E305A) had reduced [3H]-TCA efflux % (ratio of geometric means relative to OSTα/ß WT of 0.86, 0.65, and 0.79, respectively). Additionally, several OSTα/ß mutants demonstrated altered expression and cellular localization when compared with OSTα/ß WT. In summary, we identified OSTα residues (Ser228, Thr229, Gln269, Glu305) in predicted transmembrane domains that affect expression of OSTα/ß and may influence OSTα/ß-mediated bile acid transport. These data advance our understanding of OSTα/ß structure/function and can inform future studies designed to gain further insight into OSTα/ß structure or to identify additional OSTα/ß substrates and inhibitors. SIGNIFICANCE STATEMENT: OSTα/ß is a clinically important transporter involved in enterohepatic bile acid recycling with currently no high-resolution protein structure and limited structure-function data. This study identified four OSTα amino acids (Ser228, Thr229, Gln269, Glu305) that affect expression of OSTα/ß and may influence OSTα/ß-mediated bile acid transport. These data can be utilized to inform future investigation of OSTα/ß structure and refine molecular modeling approaches to facilitate the identification of substrates and/or inhibitors of OSTα/ß.

S-acylation status of bile acid transporter hASBT regulates its function, metabolic stability, membrane expression, and phosphorylation state.

The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) is the rate-limiting step of intestinal bile acid absorption in the enterohepatic circulation system of bile acids. Therefore, the regulation and stability of hASBT is vital in maintaining bile acid and cholesterol homeostasis and may serve as a potential target for cholesterol-related disorders. We hypothesized that post-translational mechanisms that govern hASBT function and regulation will provide novel insight on intestinal bile acid transport and homeostasis. In this study, we confirm the S-acylation status of hASBT via acyl biotin exchange in COS-1 cells and its impact on hASBT expression, function, kinetics, and protein stability. Using the acylation inhibitor, 2-bromopalmitate, we show that S-acylation is an important modification which modulates the function, surface expression, and maximal transporter flux (Jmax) of hASBT. By means of proteasome inhibitors, S-acylated hASBT was found to be cleared via the proteasome whereas a reduction in the palmitoylation status of hASBT resulted in rapid proteolytic degradation compared to the unmodified transporter. Screening of cysteine mutants in and or near transmembrane domains, some of which are exposed to the cytosol, confirmed Cys314 to be the predominate S-acylated residue. Lastly, we show that S-acylation was reduced in a mutant form of hASBT devoid of cytosolic facing tyrosine residues, suggestive of crosstalk between acylation and phosphorylation post-translational modification mechanisms.

Quantification of common and planar bile acids in tissues and cultured cells.

Bile acids (BAs) have been established as ubiquitous regulatory molecules implicated in a large variety of healthy and pathological processes. However, the scope of BA heterogeneity is often underrepresented in current literature. This is due in part to inadequate detection methods, which fail to distinguish the individual constituents of the BA pool. Thus, the primary aim of this study was to develop a method that would allow the simultaneous analysis of specific C24 BA species, and to apply that method to biological systems of interest. Herein, we describe the generation and validation of an LC-MS/MS assay for quantification of numerous BAs in a variety of cell systems and relevant biofluids and tissue. These studies included the first baseline level assessment for planar BAs, including allocholic acid, in cell lines, biofluids, and tissue in a nonhuman primate (NHP) laboratory animal, Macaca mulatta, in healthy conditions. These results indicate that immortalized cell lines make poor models for the study of BA synthesis and metabolism, whereas human primary hepatocytes represent a promising alternative model system. We also characterized the BA pool of M. mulatta in detail. Our results support the use of NHP models for the study of BA metabolism and pathology in lieu of murine models. Moreover, the method developed here can be applied to the study of common and planar C24 BA species in other systems.

Trends in Research and Graduate Affairs in Schools and Colleges of Pharmacy, Part 3: Underrepresented Minorities.

Objective. To examine the landscape of research and graduate affairs nationally and within schools and colleges of pharmacy. This report, part 3 of a three-part series, focuses on underrepresented minority (URM) faculty members and students, with a focus on recruitment and retention. Findings. There has been a substantial increase in recruitment of Asian faculty members by schools of pharmacy over the last 10 years, but there has been only minimal changes in the numbers of Black and Hispanic faculty numbers, which reflects the challenges in recruitment and retention of URM faculty members. Consistently low enrollment of Black and Hispanic graduate students over a 10-year period demonstrates that pharmacy schools could improve their stated diversity initiatives and goals. Despite an overall increase in PhDs conferred over the last 10 years, international students continue to receive the majority of degrees conferred. Graduation rates of Black and Hispanic students have remained low, suggesting that continued and sustained efforts are needed to recruit, support, and graduate URM students. Summary. Pharmacy schools must make a focused investment and effort toward increasing the diversity of their graduate enrollees by modeling their recruitment, enrollment, and retention strategies after national programs and best practices. Because there is a direct link between the number of faculty role models and the recruitment of students, pharmacy schools must enhance the recruitment, retention, and success of URM faculty members. Further, pharmacy schools should provide inclusion training to encourage better communication with URM advisees.

Trends in Research and Graduate Affairs in Schools and Colleges of Pharmacy, Part 2: Students.

Objective. To examine the landscape of research and graduate affairs nationally and within schools and colleges of pharmacy. This report, part 2 of a three-part series, focuses on characteristics of full-time PhD enrollees and graduates in schools and colleges of pharmacy, and career planning and preparation in graduate programs. Findings. Despite a 41% increase in funding awarded by the National Institutes of Health (NIH) to schools and colleges of pharmacy over the last 10 years, NIH funding per principal investigator only increased 14% and graduate student enrollment increased just 6% during the period. However, there was a 15% increase in PhD degrees conferred in the 10-year period, which is evidence that degree completion time decreased. The number of female graduates from pharmacy schools consistently increased, and outpaced growth in the number of male graduates by more than 10%. Most graduate programs do not include training for industry-specific skills, abilities, and experiences to better prepare graduates for nonacademic careers, although national programs have been recognized as vital to graduate student career preparation. Summary. Graduate biomedical science programs and faculty members must recognize that academia is an "alternative" career choice for their trainees, and provide job skills training to support the majority of nonacademic career choices, without compromising the rigorous training in basic biomedical disciplines.

Trends in Research and Graduate Programs in Schools and Colleges of Pharmacy, Part 1: Programs.

Objective. To examine the landscape of research and graduate education nationally and within schools and colleges of pharmacy. This report is part 1 of a three-part series and focuses on graduate programs' research funding and science faculty composition and diversity. Findings. Between FY2008 and FY2017, the number of full-time faculty members in schools and colleges of pharmacy increased 36%. The number of pharmacy schools with National Institutes of Health (NIH) awards increased by 15%, while NIH grants per faculty principal investigator (PI) increased by 31%. However, unadjusted for inflation, the mean NIH dollar amount per-faculty member PI increased just 14% and the mean NIH dollar amount per-school declined 7%, indicating that number of funded faculty outpaced dollars available. Proportionately, the percentage of science faculty members at pharmacy schools decreased from 47% to 43%. Only 15 public, research-intensive schools and colleges of pharmacy received more than half of the combined FY2017 NIH funding and total funding, while all other public and private schools and colleges of pharmacy shared the remaining funds. Interdisciplinary programs are developing slowly, and may help to diversify and increase future funding. Proportions of tenured and tenure-track positions are declining, but biological sciences and social and administrative sciences disciplines are growing and women faculty are making significant gains in these fields and at the assistant professor rank. Summary. Research-intensive schools and colleges of pharmacy are best-positioned to lead the academy to reframe graduate education to build interdisciplinary team skills and attract more diverse funding and science faculty members.

Mechanistic Insights of Phenobarbital-Mediated Activation of Human but Not Mouse Pregnane X Receptor.

Phenobarbital (PB), a broadly used antiseizure drug, was the first to be characterized as an inducer of cytochrome P450 by activation of the constitutive androstane receptor (CAR). Although PB is recognized as a conserved CAR activator among species via a well-documented indirect activation mechanism, conflicting results have been reported regarding PB regulation of the pregnane X receptor (PXR), a sister receptor of CAR, and the underlying mechanisms remain elusive. Here, we show that in a human CAR (hCAR)-knockout (KO) HepaRG cell line, PB significantly induces the expression of CYP2B6 and CYP3A4, two shared target genes of hCAR and human PXR (hPXR). In human primary hepatocytes and hCAR-KO HepaRG cells, PB-induced expression of CYP3A4 was markedly repressed by genetic knockdown or pharmacological inhibition of hPXR. Mechanistically, PB concentration dependently activates hPXR but not its mouse counterpart in cell-based luciferase assays. Mammalian two-hybrid assays demonstrated that PB selectively increases the functional interaction between the steroid receptor coactivator-1 and hPXR but not mouse PXR. Moreover, surface plasmon resonance binding affinity assay showed that PB directly binds to the ligand binding domain of hPXR (KD = 1.42 × 10-05). Structure-activity analysis further revealed that the amino acid tryptophan-299 within the ligand binding pocket of hPXR plays a key role in the agonistic binding of PB and mutation of tryptophan-299 disrupts PB activation of hPXR. Collectively, these data reveal that PB, a selective mouse CAR activator, activates both hCAR and hPXR, and provide novel mechanistic insights for PB-mediated activation of hPXR.

Tyrosine Phosphorylation Regulates Plasma Membrane Expression and Stability of the Human Bile Acid Transporter ASBT (SLC10A2).

The human apical sodium-dependent bile acid transporter (hASBT; SLC10A2) is responsible for the reclamation of bile acids from the intestinal lumen, providing a primary mechanism for bile acid and cholesterol homeostasis. However, the regulation of hASBT at the post-translational level is not well understood. In the present study, we investigated the role of Src family kinases (SFKs) and protein tyrosine phosphatases (PTPs) in the regulation of surface expression and function of hASBT. Inhibition of Src family kinases, via treatment with PP2, significantly reduced hASBT function, while the inhibition of PTPs by activated orthovanadate significantly induced function. Src family kinase inhibition by PP2 was associated with a concomitant decrease in maximum transport velocity (Jmax) correlated with a decrease in hASBT surface expression. Interestingly, PP2-mediated suppression of hASBT protein expression was rescued by the proteasome inhibitor MG132, suggesting that dephosphorylation impacts protein stability with the subsequent proteasome-dependent degradation of hASBT. Consequently, single-point mutations were introduced at five intracellular tyrosine residues: Y148F, Y216F, Y308F, Y311F, and Y337F. Although all mutants had significantly altered hASBT function without changes in total cellular expression, sequential tyrosine mutations at the five residues above rendered hASBT nonfunctional with diminished protein expression. Furthermore, orthovanadate-induced transport activity of single-point tyrosine mutants suggested a role for multiple tyrosine residues in the regulation of hASBT function and membrane expression. Overall, our data confirms that tyrosine phosphorylation mediated by Src family kinases (SFKs), in particular, regulates surface expression, function, and stability of hASBT.

Molecular Modeling of Drug-Transporter Interactions-An International Transporter Consortium Perspective.

Membrane transporters play diverse roles in the pharmacokinetics and pharmacodynamics of small-molecule drugs. Understanding the mechanisms of drug-transporter interactions at the molecular level is, therefore, essential for the design of drugs with optimal therapeutic effects. This white paper examines recent progress, applications, and challenges of molecular modeling of membrane transporters, including modeling techniques that are centered on the structures of transporter ligands, and those focusing on the structures of the transporters. The goals of this article are to illustrate current best practices and future opportunities in using molecular modeling techniques to understand and predict transporter-mediated effects on drug disposition and efficacy.Membrane transporters from the solute carrier (SLC) and ATP-binding cassette (ABC) superfamilies regulate the cellular uptake, efflux, and homeostasis of many essential nutrients and significantly impact the pharmacokinetics of drugs; further, they may provide targets for novel therapeutics as well as facilitate prodrug approaches. Because of their often broad substrate selectivity they are also implicated in many undesirable and sometimes life-threatening drug-drug interactions (DDIs).5,6.

Post-translational modifications of transporters.

Drug transporter proteins are critical to the distribution of a wide range of endogenous compounds and xenobiotics such as hormones, bile acids, peptides, lipids, sugars, and drugs. There are two classes of drug transporters- the solute carrier (SLC) transporters and ATP-binding cassette (ABC) transporters -which predominantly differ in the energy source utilized to transport substrates across a membrane barrier. Despite their hydrophobic nature and residence in the membrane bilayer, drug transporters have dynamic structures and adopt many conformations during the translocation process. Whereas there is significant literature evidence for the substrate specificity and structure-function relationship for clinically relevant drug transporters proteins, there is less of an understanding in the regulatory mechanisms that contribute to the functional expression of these proteins. Post-translational modifications have been shown to modulate drug transporter functional expression via a wide range of molecular mechanisms. These modifications commonly occur through the addition of a functional group (e.g. phosphorylation), a small protein (e.g. ubiquitination), sugar chains (e.g. glycosylation), or lipids (e.g. palmitoylation) on solvent accessible amino acid residues. These covalent additions often occur as a result of a signaling cascade and may be reversible depending on the type of modification and the intended fate of the signaling event. Here, we review the significant role in which post-translational modifications contribute to the dynamic regulation and functional consequences of SLC and ABC drug transporters and highlight recent progress in understanding their roles in transporter structure, function, and regulation.

Human bile acid transporter ASBT (SLC10A2) forms functional non-covalent homodimers and higher order oligomers.

The human apical sodium-dependent bile acid transporter, hASBT/SLC10A2, plays a central role in cholesterol homeostasis via the efficient reabsorption of bile acids from the distal ileum. hASBT has been shown to self-associate in higher order complexes, but while the functional role of endogenous cysteines has been reported, their implication in the oligomerization of hASBT remains unresolved. Here, we determined the self-association architecture of hASBT by site-directed mutagenesis combined with biochemical, immunological and functional approaches. We generated a cysteine-less form of hASBT by creating point mutations at all 13 endogenous cysteines in a stepwise manner. Although Cysless hASBT had significantly reduced function correlated with lowered surface expression, it featured an extra glycosylation site that facilitated its differentiation from wt-hASBT on immunoblots. Decreased protein expression was associated with instability and subsequent proteasome-dependent degradation of Cysless hASBT protein. Chemical cross-linking of wild-type and Cysless species revealed that hASBT exists as an active dimer and/or higher order oligomer with apparently no requirement for endogenous cysteine residues. This was further corroborated by co-immunoprecipitation of differentially tagged (HA-, Flag-) wild-type and Cysless hASBT. Finally, Cysless hASBT exhibited a dominant-negative effect when co-expressed with wild-type hASBT which validated heterodimerization/oligomerization at the functional level. Combined, our data conclusively demonstrate the functional existence of hASBT dimers and higher order oligomers irrespective of cysteine-mediated covalent bonds, thereby providing greater understanding of its topological assembly at the membrane surface.

Planar bile acids in health and disease.

Bile acids are the amphipathic primary end-products of cholesterol metabolism that aid in digestion as well as participate in signal transduction in several hepatic and enteric pathways. Despite the reputation of bile acids as signaling molecules implicated in disease states such as cancer and diabetes, there remain numerous bile acid species that are weakly characterized in either physiological or pathological conditions. This review presents one such group: the flat or planar bile acids, a set of bile acids found in humans during infancy and occurring again during certain diseases. As their name implies, these molecules are structurally distinct from the typical human bile acids, retaining the planar structure of their cholesterol predecessor instead of bending or twisting at the A ring. This review defines these species of bile acids in detail and describes their presence in infancy, gestation, and in disease. The large gaps in research regarding the flat bile acids are highlighted and all available experimental knowledge collected as far as 60years ago is summarized. Further, the potential for these molecules as endogenous biomarkers of liver disease and injury is discussed. Finally, the flat bile salts found in humans are compared to the ancestral and evolutionary older bile salts, which similarly have a flat steroidal structure, as mechanisms of flat bile acid biosynthesis are explored.

Molecular Basis of Metabolism-Mediated Conversion of PK11195 from an Antagonist to an Agonist of the Constitutive Androstane Receptor.

The constitutive androstane receptor (CAR) plays an important role in xenobiotic metabolism, energy homeostasis, and cell proliferation. Antagonism of the CAR represents a key strategy for studying its function and may have potential clinical applications. However, specific human CAR (hCAR) antagonists are limited and conflicting data on the activity of these compounds have been reported. 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a typical peripheral benzodiazepine receptor ligand, has been established as a potent hCAR deactivator in immortalized cells; whether it inhibits hCAR activity under physiologically relevant conditions remains unclear. Here, we investigated the effects of PK11195 on hCAR in metabolically competent human primary hepatocytes (HPH) and HepaRG cells. We show that although PK11195 antagonizes hCAR in HepG2 cells, it induces the expression of CYP2B6 and CYP3A4, targets of hCAR and the pregnane X receptor (PXR), in HPH, HepaRG, and PXR-knockout HepaRG cells. Utilizing a HPH-HepG2 coculture model, we demonstrate that inclusion of HPH converts PK11195 from an antagonist to an agonist of hCAR, and such conversion was attenuated by potent CYP3A4 inhibitor ketoconazole. Metabolically, we show that the N-desmethyl metabolite is responsible for PK11195-mediated hCAR activation by facilitating hCAR interaction with coactivators and enhancing hCAR nuclear translocation in HPHs. Structure-activity analysis revealed that N-demethylation alters the interaction of PK11195 with the binding pocket of hCAR to favor activation. Together, these results indicate that removal of a methyl group switches PK11195 from a potent antagonist of hCAR to an agonist in HPH and highlights the importance of physiologically relevant metabolism when attempting to define the biologic action of small molecules.

Identification of novel MRP3 inhibitors based on computational models and validation using an in vitro membrane vesicle assay.

INTRODUCTION: Multidrug resistance-associated protein 3 (MRP3), an efflux transporter on the hepatic basolateral membrane, may function as a compensatory mechanism to prevent the accumulation of anionic substrates (e.g., bile acids) in hepatocytes. Inhibition of MRP3 may disrupt bile acid homeostasis and is one hypothesized risk factor for the development of drug-induced liver injury (DILI). Therefore, identifying potential MRP3 inhibitors could help mitigate the occurrence of DILI. METHODS: Bayesian models were developed using MRP3 transporter inhibition data for 86 structurally diverse drugs. The compounds were split into training and test sets of 57 and 29 compounds, respectively, and six models were generated based on distinct inhibition thresholds and molecular fingerprint methods. The six Bayesian models were validated against the test set and the model with the highest accuracy was utilized for a virtual screen of 1470 FDA-approved drugs from DrugBank. Compounds that were predicted to be inhibitors were selected for in vitro validation. The ability of these compounds to inhibit MRP3 transport at a concentration of 100µM was measured in membrane vesicles derived from stably transfected MRP3-over-expressing HEK-293 cells with [3H]-estradiol-17ß-d-glucuronide (E217G; 10µM; 5min uptake) as the probe substrate. RESULTS: A predictive Bayesian model was developed with a sensitivity of 73% and specificity of 71% against the test set used to evaluate the six models. The area under the Receiver Operating Characteristic (ROC) curve was 0.710 against the test set. The final selected model was based on compounds that inhibited substrate transport by at least 50% compared to the negative control, and functional-class fingerprints (FCFP) with a circular diameter of six atoms, in addition to one-dimensional physicochemical properties. The in vitro screening of predicted inhibitors and non-inhibitors resulted in similar model performance with a sensitivity of 64% and specificity of 70%. The strongest inhibitors of MRP3-mediated E217G transport were fidaxomicin, suramin, and dronedarone. Kinetic assessment revealed that fidaxomicin was the most potent of these inhibitors (IC50=1.83±0.46µM). Suramin and dronedarone exhibited IC50 values of 3.33±0.41 and 47.44±4.41µM, respectively. CONCLUSION: Bayesian models are a useful screening approach to identify potential inhibitors of transport proteins. Novel MRP3 inhibitors were identified by virtual screening using the selected Bayesian model, and MRP3 inhibition was confirmed by an in vitro transporter inhibition assay. Information generated using this modeling approach may be valuable in predicting the potential for DILI and/or MRP3-mediated drug-drug interactions.

Potent and Selective Inhibition of Plasma Membrane Monoamine Transporter by HIV Protease Inhibitors.

Plasma membrane monoamine transporter (PMAT) is a major uptake-2 monoamine transporter that shares extensive substrate and inhibitor overlap with organic cation transporters 1-3 (OCT1-3). Currently, there are no PMAT-specific inhibitors available that can be used in in vitro and in vivo studies to differentiate between PMAT and OCT activities. In this study, we showed that IDT307 (4-(4-(dimethylamino)phenyl)-1-methylpyridinium iodide), a fluorescent analog of 1-methyl-4-phenylpyridinium (MPP+), is a transportable substrate for PMAT and that IDT307-based fluorescence assay can be used to rapidly identify and characterize PMAT inhibitors. Using the fluorescent substrate-based assays, we analyzed the interactions of eight human immunodeficiency virus (HIV) protease inhibitors (PIs) with human PMAT and OCT1-3 in human embryonic kidney 293 (HEK293) cells stably transfected with individual transporters. Our data revealed that PMAT and OCTs exhibit distinct sensitivity and inhibition patterns toward HIV PIs. PMAT is most sensitive to PI inhibition whereas OCT2 and OCT3 are resistant. OCT1 showed an intermediate sensitivity and a distinct inhibition profile from PMAT. Importantly, lopinavir is a potent PMAT inhibitor and exhibited >120 fold selectivity toward PMAT (IC50 = 1.4 ± 0.2 µM) over OCT1 (IC50 = 174 ± 40 µM). Lopinavir has no inhibitory effect on OCT2 or OCT3 at maximal tested concentrations. Lopinavir also exhibited no or much weaker interactions with uptake-1 monoamine transporters. Together, our results reveal that PMAT and OCTs have distinct specificity exemplified by their differential interaction with HIV PIs. Further, we demonstrate that lopinavir can be used as a selective PMAT inhibitor to differentiate PMAT-mediated monoamine and organic cation transport from those mediated by OCT1-3.

Internalization and Subcellular Trafficking of Poly-l-lysine Dendrimers Are Impacted by the Site of Fluorophore Conjugation.

Internalization and intracellular trafficking of dendrimer-drug conjugates play an important role in achieving successful drug delivery. In this study, we aimed to elucidate the endocytosis mechanisms and subcellular localization of poly-l-lysine (PLL) dendrimers in Caco-2 cells. We also investigated the impact of fluorophore conjugation on cytotoxicity, uptake, and transepithelial transport. Oregon green 514 (OG) was conjugated to PLL G3 at either the dendrimer periphery or the core. Chemical inhibitors of clathrin-, caveolin-, cholesterol-, and dynamin-mediated endocytosis pathways and macropinocytosis were employed to establish internalization mechanisms, while colocalization with subcellular markers was used to determine dendrimer trafficking. Cell viability, internalization, and uptake were all influenced by the site of fluorophore conjugation. Uptake was found to be highly dependent on cholesterol- and dynamin-mediated endocytosis as well as macropinocytosis. Dendrimers were trafficked to endosomes and lysosomes, and subcellular localization was impacted by the fluorophore conjugation site. The results of this study indicate that PLL dendrimers exploit multiple pathways for cellular entry, and internalization and trafficking can be impacted by conjugation. Therefore, design of dendrimer-drug conjugates requires careful consideration to achieve successful drug delivery.