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. Author manuscript; available in PMC: 2021 May 20.
Published in final edited form as: Bioconjug Chem. 2020 Apr 10;31(5):1344–1353. doi: 10.1021/acs.bioconjchem.0c00079

Anionic Polymers Promote Mitochondrial Targeting of Delocalized Lipophilic Cations

Ziwen Jiang †, Hongxu Liu †, Huan He †, Nagendra Yadava ‡,§, James J Chambers ∥, S Thayumanavan †,§,∥,*
PMCID: PMC7347245  NIHMSID: NIHMS1605679  PMID: 32208679

Abstract

Mitochondria are therapeutic targets in many diseases including cancer, metabolic disorders, and neurodegenerative diseases. Therefore, strategies to deliver therapeutics of interest to mitochondria are important for therapeutic development. As delocalized lipophilic cations (DLCs) preferentially accumulate into mitochondria, DLC-conjugation has been utilized facilitate therapeutic delivery systems with mitochondrial targeting capability. Here we report that upon DLC-conjugation, anionic polymers exhibited significantly improved mitochondrial targeting when compared to cationic polymers and charge-neutral polymers. Considering that cell membrane generally bears net negative charge, the observed phenomenon is unexpected. Notably, the DLC-conjugated anionic polymers circumvented the endosomal entrapment. The rapid mitochondrial accumulation of DLC-conjugated anionic polymers is likely a membrane-potential driven process, along with the involvement of the mitochondrial pyruvate carrier. Moreover, the structural variations on the side chain of DLC-conjugated anionic polymers did not compromise the overall mitochondrial targeting capability, widely extending the applicability of anionic macromolecules in therapeutic delivery systems.

Keywords: Anionic polymer, delocalized lipophilic cation, conjugation, mitochondrial targeting

Graphical Abstract

graphic file with name nihms-1605679-f0001.jpg

Introduction

The mitochondrion is the central organelle for generating adenosine triphosphate (ATP), supplying requisite energy for cellular metabolism.1, 2 Mitochondrial dysfunction leads to cellular disorders and eventually causes severe diseases such as neurodegeneration.3 Beyond the role of cellular powerhouse, mitochondria are also involved in the regulation of the intrinsic apoptotic pathway.4 Briefly, mitochondria control the translocation of pro-apoptotic proteins to regulate the activation of apoptotic effectors (e.g. caspases).5 Defects in the apoptotic pathways have been associated with the resistance of tumor cells towards chemotherapy,6 further implying the fundamental importance of mitochondria as therapeutic targets. Besides, mitochondria are central mediators in regulated necrosis.7 Thus, strategies for precisely delivering drugs to mitochondria are necessary for the development of mitochondria-relevant therapeutics.

Chemical approaches to targeting mitochondria are mainly achieved through delocalized lipophilic cations (DLCs) or signal peptides.8 DLCs are positively charged small molecules, possessing delocalized electronic structures via resonance stabilization of the lipophilic molecule.9 The positive charge within DLCs is spread over a large hydrophobic molecular area, therefore requiring a lower enthalpy input to desolvate these charged species. As a result, moving DLCs through lipid bilayers requires a far lower activation energy than hydrophilic cations such as Na+.10 Driven by the cell and mitochondrial membrane potentials, DLCs readily transfer into the cytosol and efficiently accumulate to the mitochondria.8 Representative DLCs include triphenylphosphonium-based compounds,11 rhodamine-12 and cyanine (Cy)-based13 derivatives.

DLC-conjugation has been utilized to initiate the mitochondrial targeting capability of therapeutic delivery systems.9, 14-17 While the DLC-conjugation effectively targets small molecules to mitochondria, the DLC-conjugation with large and polar molecules has been far less effective in their mitochondrial targeting.18, 19 Herein, we report that conjugating DLCs onto anionic polymers resulted in a rapid mitochondrial targeting effect, largely exceeding the cationic and charge-neutral polymers with DLC-conjugation. Specifically, conjugation with anionic polymers did not compromise the mitochondrial targeting capability of delocalized lipophilic cations. To explain such unexpected phenomenon, we conducted structural variations on the DLC-conjugated anionic polymers, extending the fundamental understanding on the design principles of mitochondrial targeting materials. The study presents an effective strategy for enhancing the cellular uptake and mitochondrial targeting capability of therapeutic delivery systems.

Results and Discussion

Cy3-conjugation on anionic polymers enhanced their cellular uptake

The molecular design of polymers allows an azide group displayed as the end group of each polymer chain, providing the reaction site for further functionalization with click reaction.20 First, a library of methacrylate polymers with different surface charges were synthesized using reversible addition-fragmentation chain-transfer (RAFT) polymerization (Figure 1a). Next, dibenzocyclooctyne-functionalized fluorescent molecules were covalently conjugated to the azide-tagged polymers to allow their intracellular tracking (Figure S1). During the evaluation of cellular uptake, we fortuitously discovered that the Cyanine 3 (Cy3)-conjugated anionic polymers (NEG or SO3) exhibited a significantly higher uptake efficiency than the rest of the structural analog, including a cationic polymer (POS) and two charge-neutral polymers (PEG and MPC) (Figure S2). To further validate this phenomenon, we extended the screening to three more cell types, including human umbilical vein endothelial cells (HUVECs), mesenchymal stem cells immortalized with hTERT (hTERT-MSC), and mouse myoblast cells (C2C12). The results from these three cell types are all in agreement with the results from human cervical cancer cells (HeLa) (Figure 1b, S3), confirming that the Cy3-conjugated anionic polymers exhibit an enhanced cellular uptake compared to other polymers with different surface charge.

Figure 1.

Figure 1.

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(a) Chemical structure of Cy3-labelled amphiphilic polymers with different surface charge. (b) Cellular uptake of Cy3-labelled amphiphilic polymers in different cell types. Cy3-tagged polymers were shown in red and Hoechst 333429-stained nucleus was shown in blue. Scale bar represents 20 μm.

Cy3-conjugated anionic polymers localized on the mitochondria

Next, we explored the intracellular localization of the Cy3-labeled anionic polymer (NEG). From the colocalization assessment with commercially available organelle-staining reagents, we found that NEG highly overlapped with the mitochondrial stain, rather than the lysosome and endoplasmic reticulum (ER) stains (Figure 2, S4). The colocalization analysis of ER stain and NEG exhibited the colocalization coefficient at ~0.59 in HeLa cells, and the coefficient decreased to ~0.35 in SK-MEL-2 cells (Figure S5), further proving that ER is less likely to be the localization of NEG. Since the ER-mitochondria contact sites closely correlate with the cellular metabolism,21, 22 the difference of ER stain-NEG colocalization coefficient can be potentially attributed to varied metabolic activities among different cell lines.23

Figure 2.

Figure 2.

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Cy3-tagged anionic polymers (NEG) localize to the mitochondria of HeLa cells rather than the lysosomes and endoplasmic reticulum. Corresponding organelle trackers (LysoTracker for lysosomes, ER-Tracker for endoplasmic reticulum, and MitoTracker for mitochondria) were shown in green. MitoGFP stands for HeLa cells with green fluorescence proteins transiently expressed within their mitochondria (shown in green). Cy3-tagged polymers were shown in red. Scale bar represents 10 μm. The colocalization analysis at the bottom of each column was performed by comparing the green and red color intensity of each pixel in the merged image (r stands for the Pearson’s correlation coefficient). The MitoGFP group has lower r value than the MitoTracker Green group, possibly due to the transient expression nature of the green fluorescence proteins.

To further validate the colocalization results with small molecule-based staining, we employed HeLa cells with green fluorescence proteins transiently expressed within the mitochondria (MitoGFP). After incubating Cy3-labeled anionic polymers with the MitoGFP-expressing HeLa cells, we observed similar colocalization results throughout the tubular morphology as with the small molecule-based mitochondrial stain (MitoTracker Green), confirming that Cy3-labeled anionic polymers are preferentially localized to mitochondria. Since the expression of MitoGFP is transient, it is reasonable to observe a lower colocalization coefficient (~0.57) than the MitoTracker Green group (~0.85). Note that NEG bypassed endosomal entrapment during its cellular uptake process. In contrast, Cy3-labeled cationic polymers (POS) were found to be mainly accumulated in the endosomes (Figure S6). Previous results have also demonstrated that DLC-conjugated cationic materials accumulate in endosomes and do not efficiently reach mitochondria.18

Cy3-conjugation lead to the mitochondrial targeting of anionic polymers

The enhanced cellular uptake and mitochondrial targeting capability of Cy3-labeled anionic polymer might be attributed to the conjugation of Cy3, as Cy3 itself is a delocalized lipophilic cation (DLC).24, 25 The azide-tagged end group allows a facile variation on the dye to be conjugated on the polymer, i.e. any azide-reactive dye derivatives can be tagged on the polymer. To test our hypothesis, we functionalized the azide-tagged anionic polymers (NEG) with five different dye molecules (Figure S7). The cellular uptake results from anionic polymers conjugated with different dyes altogether revealed that the conjugated dye molecule indeed determines the mitochondrial targeting capability of anionic polymers (Figure 3, S8). Polymers conjugated with DLC exhibited precise localization on mitochondria, whereas conjugation with non-DLC dye mostly resulted in the endosomal entrapment. Particularly regarding the sulfonated Cy3 (Sulfo-Cy3) analog, where the original DLC structure of Cy3 is altered to an overall charge-neutral dye with the sulfonate substitution, eventually resulting in the loss of overall mitochondrial targeting of anionic polymers. Note that the anionic polymer itself is based on sulfonate moieties, but only when installing this functionality in Cy3 itself that mutes the mitochondrial targeting capability. Also, based on the structural design of the polymer, introducing mitochondrial targeting capability to anionic polymers only requires one targeting moiety per polymer chain.

Figure 3.

Figure 3.

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(a) Reaction between azide-terminated polymers and DBCO-based dye. (b) Cellular uptake of anionic polymers with different dye conjugated at the polymeric chain end. Conjugation with delocalized lipophilic cations (DLCs) localized anionic polymers to the mitochondria of HeLa cells (Cy3- and Cy7-conjugation), whereas non-DLC conjugated anionic polymers did not reach mitochondria (MB 488- and Sulfo-Cy3-conjugation). Hoechst 333429-stained nucleus was shown in blue. Scale bar represents 20 μm.

The uptake of Cy3-conjugated anionic polymer is membrane potential-dependent

Efficient mitochondrial targeting of these Cy3-conjugated anionic polymers was observed within an hour through time-lapse microscopic imaging (Video S1). Considering the anionic feature of NEG, their cellular uptake and mitochondrial targeting may be contributed by the cell surface receptors that recognize anionic macromolecules.26 After treating HeLa cells with dextran sulfate or polyinosinic acid (Figure 4a,b), two inhibitors for scavenger receptors,27 the cellular uptake of NEG was not affected. Moreover, inhibition of mitochondrial voltage-dependent anion channels using erastin28 did not reduce the uptake of NEG (Figure 4c). The increase of NEG uptake after erastin-treatment is possibly due to the alteration of mitochondrial membrane permeability.28 Nonetheless, the role of DLC for the uptake of NEG is worthy of investigation. Since the uptake of DLC is known to be driven by mitochondrial membrane potential (ΔΨm), we treated cells with either FCCP or oligomycin to modulate the ΔΨm towards two different directions.29 As a result, FCCP-treatment decreased the uptake of NEG while oligomycin-treatment increased the uptake of NEG, confirming that the uptake is driven by membrane potential (Figure 4d,e). Meanwhile, we also evaluated the cellular uptake of NEG after treating cells with UK 5099, a potent inhibitor for the plasma membrane monocarboxylate transporters and the mitochondrial pyruvate carrier.30, 31 The role of mitochondrial pyruvate carrier during the uptake of Cy3-labeled anionic polymers was later evaluated by depleting sodium pyruvate within cell culture medium (Figure S13). The decrease of NEG uptake upon increased dosage of UK 5099-treatment indicates the participation of these transporters during the cellular uptake of NEG (Figure 4f). Based on our previous results, the cellular uptake of NEG also involves macropinocytosis.20

Figure 4.

Figure 4.

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Effect of pharmacological inhibitors on the cellular uptake of Cy3-tagged anionic polymers in HeLa cells. Error bars in each figure represent the standard deviation of four replicates.

Side chain variations on Cy3-conjugated polymers did not affect the mitochondrial targeting

It is important to understand the effect of side chain structure on the mitochondrial targeting capability of anionic polymers. NEG is a random copolymer containing three side chain moieties, including sulfonate derivatives to ensure the anionic feature, poly(ethylene glycol) derivatives as the hydrophilic moieties, and pyridyl disulfide (PDS) derivatives as the hydrophobic moieties. We first varied the structure of the hydrophobic side chain and assessed if the mitochondrial targeting ability of these polymers are affected. It turns out that modulating either the disulfide bond (PDB) or the pyridine structure (Ph and C4S2) do not alter the mitochondrial targeting of Cy3-conjugated anionic polymers. Moreover, after replacing the PDS side chain with either hydrophobic hexyl (C6) or hydrophilic hydroxyl (OH) group, mitochondrial localization of polymers was still observed from 13 different cell types (Figure 5, S9).

Figure 5.

Figure 5.

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(a) Chemical structure of Cy3-tagged anionic polymers with varied side chains. (b) Mitochondrial localization of Cy3-conjugated polymers with different side chains in different cell types. Scale bar represents 10 μm.

The effect of charge density and negative charge type were also investigated regarding the Cy3-conjugated anionic polymers. Such Cy3-conjugated anionic polymers with varied density of sulfonate moieties again exhibited strong colocalization with mitochondrial staining, revealing that the mitochondrial targeting capability is independent of the negative charge density (Figure S10). We also synthesized an analog of anionic polymers with different negative charge type, containing either carboxylate (COOH) or phosphate (PO4) instead of sulfonate (SO3). After conjugating these polymers respectively with Cy3, similarly efficient mitochondrial targeting was observed from SO3 and PO4, while COOH did not present such efficiency (Figure 6, S12).

Figure 6.

Figure 6.

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(a) Chemical structure of Cy3-tagged anionic polymers with varied negatively charged groups. (b) Mitochondrial localization of Cy3-conjugated polymers with different anionic side chains in different cell types. Scale bar represents 10 μm.

The results from pharmacological inhibitor screening indicates the role of plasma membrane monocarboxylate transporter and mitochondrial pyruvate carrier during the uptake of these Cy3-labeled anionic polymers (Figure 4f). Considering the pyruvate and carboxylate species (such as sodium pyruvate and linoleic acid) within the cell culture medium, the suppressed cellular uptake of COOH can be attributed to the competition with these small molecules (Figure S13). While we were preparing the current manuscript, a report in press demonstrated that conjugating carboxylate-decorated polymers with Cy5 targeted the polymers to the mitochondria of rat neural cells.32 In our systems, we found that COOH indeed can enhance mitochondrial targeting in two out of the nine cell lines tested (Myotube and SK-MEL-2; see Figure 6). We found however that sulfonates and phosphates have more consistent mitochondrial targeting capability. Interestingly, as nucleic acids similarly contain phosphate groups as PO4, conjugating DLC with nucleic acids is expected to efficiently localize nucleic acids to mitochondria. Such effect has been successfully demonstrated in a previous report,33 supporting our hypothesis.

Cy3-conjugation on anionic polymers enhanced the small-molecule drug delivery efficiency of anionic polymers

As DLC-conjugated anionic polymers possess efficient mitochondrial targeting capability, DLC-conjugated anionic polymers are expected to have elevated delivery efficiency as drug carriers. We employed PDB as a drug carrier for small-molecule drugs (doxorubicin, lonidamine, and gossypol) to test our hypothesis. In detail, small-molecule drug of interest was encapsulated into the anionic polymer-based micelles respectively before and after Cy3-conjugation (PDB w/o Cy3 and PDB) (Figure S14). Resazurin-based assay was applied to evaluate the drug delivery efficiency.34 Resazurin is a redox-sensitive fluorogenic substrate. Once resazurin is reduced to resorufin (highly fluorescent) in mitochondria, relative level of the reduction reaction product reflects the mitochondrial metabolic activity.

PDB loaded with doxorubicin (IC50 = 5.0 μM), is more effective than doxorubicin alone (IC50 = 6.0 μM) to inhibit the mitochondrial metabolic activity (Figure 7a). Other than the well-known mechanism of action via the intercalation of DNA,35 doxorubicin has been associated with mitochondrial dysfunctions.36 In contrast, the therapeutic effect of doxorubicin was suppressed after being encapsulated inside PDB w/o Cy3. This is reasonable since anionic polymers without DLC-conjugation tends to be entrapped in the endosomes (Figure 3b), limiting the drug efficacy. Other than doxorubicin, similar trend was observed from two small-molecule drugs with mitochondrial proteins as the drug target, where lonidamine inhibits mitochondrial hexokinase37 and gossypol inhibits Bcl-2 family proteins as apoptosis regulator (Figure 7b,c).38 Note that before loading, both PDB w/o Cy3 and PDB carriers at each dosage demonstrated negligible effect on the metabolic activity of mitochondria in HeLa cells (Figure 7d). These datasets demonstrated that the DLC-conjugation enhances the drug delivery efficiency of anionic polymers. Meanwhile, such platform is a widely applicable for targeting small-molecule drugs to mitochondria, circumventing specific chemical modifications on the drug of interest.

Figure 7.

Figure 7.

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Cy3-tagged anionic polymers (PDB) demonstrated enhanced drug delivery efficiency than anionic polymers without Cy3 conjugated (PDB w/o Cy3). Small molecule-based drugs including (a) Doxorubicin, (b) Lonidamine, and (c) Gossypol were respectively encapsulated into either PDB or PDB w/o Cy3. After incubating with drug-loaded polymers or drug only control for 24 hours with HeLa cells, the mitochondrial metabolic activity of treated cells was evaluated by the resorufin intensity through alamarBlue assay. Error bars in each figure represent the standard deviation of four replicates.

Conclusions

In summary, we describe the conjugation of delocalized lipophilic cations (DLCs) on anionic polymers to facilitate macromolecules with efficient mitochondrial targeting capability. Unlike the endosomal entrapment of DLC-conjugated cationic or charge-neutral polymers, DLC-conjugated anionic polymers rapidly and efficiently localize to the mitochondria of live cells. Apart from membrane potential as the driving force, the mitochondrial targeting process of DLC-conjugated anionic polymers is also attributed to the mitochondrial pyruvate carrier proteins. Moreover, upon DLC-conjugation, the anionic polymer library with varied amphiphilicity and charge density successfully target at the mitochondria, providing fundamental understandings on the design principles of mitochondrial targeting materials. The efficient mitochondrial targeting capability clearly requires the co-existence of DLC and anionic polymeric chain, as replacing either component largely impairs such capability. While we attribute the membrane potential and mitochondrial pyruvate carrier proteins as key players in this targeting process, further understanding of the features that underlie this phenomenon still needs investigation, including elucidating the localization of these polymers in specific mitochondrial sub-compartments. Meanwhile, the pH gradient within mitochondria could also play a role in the mitochondrial localization of these polymers.39 From applications perspective, the versatility in the design of anionic macromolecules opens up potential avenues in biological imaging and therapeutic delivery applications.

Experimental Procedures

General methods.

Reagents were purchased from commercial sources without further purification. 1H NMR, 13C NMR, and 31P NMR spectra were recorded from a Bruker AdvanceIII 400 (or 500) NMR spectrometer. When using tetrahydrofuran (THF) as the eluent, gel permeation chromatography (GPC) was performed on an Agilent 1260 LC. Molecular weights are versus polystyrene standards. When using trifluoroethanol (TFE) as the eluent, GPC was performed on an Agilent 1200 series HPLC system, using polymethylmethacrylate standards for molecular weight calculation. Dynamic light scattering and zeta potential measurement were measured on a Malvern Zetasizer Nano ZS. Confocal microscopy images were acquired from a Nikon fluorescence microscope equipped with a spectral detector unit. Confocal microscopy video was obtained from a Nikon fluorescence microscope equipped with a Yokogawa spinning disk unit. Flow cytometry experiments were conducted on a ThermoFisher Attune NxT flow cytometer. The infrared spectra were obtained on a Bruker Alpha FT-IR Spectrometer with a spectral range from 3500 cm−1 to 400 cm−1. Thermogravimetric analysis was conducted under N2 flow from room temperature to 600 °C using a TA Instrument Q50 thermogravimetric analyzer.

Synthesis and characterization of polymers.

Polymers in this study were all synthesized using reversible addition–fragmentation chain-transfer polymerization. Azido-derivatives of chain transfer agent and radical initiators were synthesized based on a previous report.20 Detailed procedures for the synthesis and characterization results of monomers and polymers are summarized in Section 2.1., 2.2., and Section 3. of the Supplementary Information. The conjugation between azide-tagged polymers and DBCO-derivatives was carried out based on our previous report.20 Details are available in Section 2.3. of the Supplementary Information. The stock solution of each polymer was prepared in deionized water (details in Section 2.4. of the Supplementary Information).

Cellular uptake of polymers with different surface charge or different dye conjugation.

General cell procedures are summarized in Section 2.5 of the Supplementary Information. Cells of interest were seeded into a glass bottom dish (Cellvis, #D35C4-20-0-N) prior to the experiment. Next, the cells were incubated with Cy3-labelled polymers (30 μg·mL−1 for PEG, POS, NEG, and MPC in full growth medium) for 18 hours. After washing with phosphate buffer saline, the cells were incubated with Hoechst 33342 in FluoroBrite DMEM at 37 °C to stain the nucleus. The intracellular distribution was measured by confocal microscopy with excitation wavelengths of 405 nm (Hoechst 33342) and 561 nm (Cy3-labelled polymers). Details on each cell type are available in Section 2.6. and 2.8. of the Supplementary Information.

Subcellular localization of Cy3-tagged anionic polymers.

A total of 50 k HeLa cells were seeded into a glass bottom dish (Cellvis, #D35C4-20-0-N) for 24 hours prior to the experiment. Subsequently, the cells were incubated with Cy3-labelled anionic polymers (NEG, 60 μg·mL−1 in DMEM) for 2 hours. Before staining cells with organelle trackers, cells were washed with phosphate buffer saline. For organelle staining, the cells were incubated with LysoTracker Green, ER-Tracker Green, and MitoTracker Green were respectively used to stain the cellular lysosomes, endoplasmic reticulum (ER), and mitochondria. The mitochondria colocalization was further evaluated in HeLa cells with green fluorescence proteins transiently expressed within the mitochondria. The intracellular distribution was measured by confocal microscopy. Experimental details along with the colocalization analysis are available in Section 2.7. of the Supplementary Information.

Effect of pharmacological inhibitors.

A total of 10 k HeLa cells were cultured in a 96-well plate for 24 hours prior to the experiment. Next, cells were cultured for 1 hour in DMEM containing dextran sulfate polyinosinic acid, erastin, FCCP, oligomycin, UK 5099, respectively. Subsequently, in the presence of inhibitors, the cells were incubated with Cy3-labelled anionic polymers (NEG, 2 μg·mL−1 in DMEM) for additional 2 hours. After washing the cells with cold PBS, the fluorescence intensity of Cy3 within the cells was measured using flow cytometry with an excitation wavelength of 561 nm. For the positive control, HeLa cells were cultured in DMEM for 1 hour and incubated with Cy3-labelled polymers for another 2 hours. For the blank group, HeLa cells were cultured in DMEM for 3 hours. After subtracting the fluorescence signal from the blank group, Cy3 fluorescence of the positive control group was normalized as 100%. Details are available in Section 2.9. of the Supplementary Information.

Cellular uptake of anionic polymers with varied side chains or different charge densities.

A total of 50 k A549 cells were seeded into a glass bottom dish (Cellvis, #D35C4-20-0-N) for 24 hours prior to the experiment. Next, the cells were incubated with Cy3-labelled polymers in respective growth medium for 2 hours. Details are available in Section 2.10. and 2.11. of the Supplementary Information. After washing with phosphate buffer saline, the cells were maintained in FluoroBrite DMEM for confocal microscopic measurement. The intracellular distribution of Cy3-labelled polymers was measured by confocal microscopy with an excitation wavelength of 488 nm. Multispectral images were collected in each run to record with emission wavelengths from 492 nm to 682 nm, obtaining 32 images in total (5-nm wavelength each). Images with emission wavelengths from 560 nm to 630 nm were combined as the final intracellular distribution of Cy3-labelled polymers.

Delivery of small-molecule drugs using Cy3-conjugated anionic polymers.

Polymer was dissolved in trifluoroethanol, followed by the dropwise addition of deionized water while stirring. The stock solution of small molecule-drug of interest in DMSO was added into the solution while stirring. The mixture was continuously stirred at room temperature overnight. Next, the mixture was purified with deionized water and concentrated using Amicon centrifugal filters with 3 k MWCO. With the assumption of no sample loss during the purification, the volume of the concentrated polymer solution was adjusted to result in the polymeric micelle stock solution at a concentration of 10 mg·mL−1, containing 0.5 mg·mL−1 small-molecule drug of interest. Empty polymeric micelles as the control groups were prepared similarly without the drug loading step. Mitochondrial metabolic activity of cells after delivery was evaluated using alamarBlue assay. Details are available in Section 2.12. of the Supplementary Information.

Supplementary Material

Supporting Information
VideoS1
Download video file (5.6MB, mp4)

Acknowledgements

We thank the NIGMS of the National Institutes of Health (GM-128181) for support. We are grateful to the colleagues at UMass Amherst for kindly providing the following cells: BT549 and hTERT-MSC cells from Dr. Shelly R. Peyton, C2C12 cells from Dr. Lawrence M. Schwartz, primary mouse embryo fibroblasts from Dr. Jesse Mager, COS-1 cells from Dr. Vincent M. Rotello, HepG2 cells from Dr. R. Thomas Zoeller, and Jurkat cells from Dr. Barbara A. Osborne. We thank Dr. Mingxu You (UMass Amherst) for kindly sharing the flow cytometer. Microscopy data collection was performed in the Light Microscopy Facility and Nikon Center of Excellence at the Institute for Applied Life Sciences, University of Massachusetts Amherst with support from the Massachusetts Life Science Center.

Footnotes

The authors declare no competing financial interest.

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website.

Detailed experimental section, characterization data, and supporting figures.

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