Controlling Cell Organization in 3D Coculture Spheroids Using DNA Interactions.

The role of stromal and immune cells in transforming the tumor microenvironment is a key consideration in understanding tumor cell behavior and anticancer drug development. To better model these systems in vitro, 3D coculture tumor spheroids have been engineered using a variety of techniques including centrifugation to microwells, hanging drop, low adhesion cultures, and culture of cells in a microfluidic platform. Aside from using bioprinting, however, it has remained more challenging to direct the spatial organization of heterotypic cells in standalone 3D spheroids. To address this, we present an in vitro 3D coculture tumor model where we modulated the interactions between cancer cells and fibroblasts through DNA hybridization. When native heterotypic cells are simply mixed, the cell aggregates typically show cell sorting behavior to form phase separated structures composed of single cell types. In this work, we demonstrate that when MDA-MB-468 breast cancer and NIH/3T3 fibroblasts are directed to associate via complementary DNA, a uniform distribution of the two cell types within a single spheroid was observed. In contrast, in the absence of specific DNA interactions between the cancer cells and fibroblasts, individual clusters of the NIH/3T3 cells formed in each spheroid due to cell sorting. To better understand the effect of heterotypic cell organization on either cell-cell contacts or matrix protein production, the spheroids were further stained with anti-E-cadherin and antifibronectin antibodies. While the amounts of E-cadherin appeared to be similar between the spheroids, a significantly higher amount of fibronectin secretion was observed in the coculture spheroids with uniform mixing of two cell types. This result showed that different heterotypic cell distributions within 3D architecture can influence the ECM protein production that can again alter the properties of the tumor or tumor microenvironment. The present study thus describes the use of DNA templating to direct the organization of cells in coculture spheroids, which can provide mechanistic biological insight into how heterotypic distribution in tumor spheroids can influence tumor progression, metastasis, and drug resistance.

Silica-coated gold nanorods with hydrophobic modification show both enhanced two-photon fluorescence and ultrasound drug release.

Hydrophobically-modified silica-coated gold nanorods are presented here as multifunctional theranostic agents. A single modification both increases two-photon fluorescence and promotes cavitation-based acoustic signal for imaging. A two-fold greater release of small molecule drugs was observed under ultrasound-mediated conditions as compared to passive release without ultrasound.

Enzyme Prodrug Therapy with Photo-Cross-Linkable Anti-EGFR Affibodies Conjugated to Upconverting Nanoparticles.

In this work, we demonstrate that a photo-cross-linkable conjugate of upconverting nanoparticles and cytosine deaminase can catalyze prodrug conversion specifically at tumor sites in vivo. Non-covalent association of proteins and peptides with cellular surfaces leads to receptor-mediated endocytosis and catabolic degradation. Recently, we showed that covalent attachment of proteins such as affibodies to cell receptors yields extended expression on cell surfaces with preservation of protein function. To adapt this technology for in vivo applications, conjugates were prepared from upconverting nanoparticles and fusion proteins of affibody and cytosine deaminase enzyme (UC-ACD). The affibody allows covalent photo-cross-linking to epidermal growth factor receptors (EGFRs) overexpressed on Caco-2 human colorectal cancer cells under near-infrared (NIR) light. Once bound, the cytosine deaminase portion of the fusion protein converts the prodrug 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU). NIR covalent photoconjugation of UC-ACD to Caco-2 cells showed 4-fold higher retention than observed with cells that were not irradiated in vitro. Next, athymic mice expressing Caco-2 tumors showed 5-fold greater UC-ACD accumulation in the tumors than either conjugates without the CD enzyme or UC-ACDs in the absence of NIR excitation. With oral administration of 5-FC prodrug, tumors with photoconjugated UC-ACD yielded 2-fold slower growth than control groups, and median mouse survival increased from 28 days to 35 days. These experiments demonstrate that enzyme-decorated nanoparticles can remain viable after a single covalent photoconjugation in vivo, which can in turn localize prodrug conversion to tumor sites for multiple weeks.

Effect of covalent photoconjugation of affibodies to epidermal growth factor receptor (EGFR) on cellular quiescence.

Cellular quiescence is a reversible state of cell cycle arrest whereby cells are temporarily maintained in the nondividing phase. Inducing quiescence in cancer cells by targeting growth receptors is a treatment strategy to slow cell growth in certain aggressive tumors, which in turn increases the efficacy of treatments such as surgery or systemic chemotherapy. However, ligand interactions with cell receptors induce receptor-mediated endocytosis followed by proteolytic degradation, which limits the duration of cellular quiescence. Here, we report the effects of targeted covalent affibody photoconjugation to epidermal growth factor receptors (EGFR) on EGFR-positive MDA-MB-468 breast cancer cells. First, covalently conjugating affibodies to cells increased doubling time two-fold and reduced ERK activity by 30% as compared to cells treated with an FDA-approved anti-EGFR antibody Cetuximab, which binds to EGFR noncovalently. The distribution of cells in each phase of the cell cycle was determined, and cells conjugated with the affibody demonstrated an accumulation in the G1 phase, indicative of G1 cell cycle arrest. Finally, the proliferative capacity of the cells was determined by the incorporation of 5-ethynyl-2-deoxyuridine and Ki67 Elisa assay, which showed that the percentage of proliferative cells with photoconjugated affibody was half of that found for the untreated control.

Generation of 3D cellular spheroids using DNA modified cell receptors and programmable DNA interactions.

3D culture is known to provide more faithful tissue models than 2D culture, and thus it is a valuable tool for in vitro evaluation of biological models. However, many cell lines are unable to form desired 3D spheroids by traditional methods because the naturally occurring cell-cell adhesion is too weak. Here, we present a method to produce 3D cell spheroids by using DNA-mediated assembly. We first demonstrate an Affinity Mediated Photoconjugation Approach (AMCP) to covalently modify cell receptors with affibody-streptavidin fusion proteins, where the affibody chemically crosslinks to cell expressed EGFR and the streptavidin is used to attach DNA strands. The DNA conjugated cells were then mixed with complementary DNA 'linker strands' to impart cell-cell interactions. When incubated in wells coated with non-adhesive polymers, cells formed dense spherical aggregates larger than 500 microns in diameter. Each of these studies was carried out using human breast cancer cells (MBA-MB-468), aneuploid human keratinocytes (HaCaT), and human colon cancer cells (Caco-2). Without either DNA on the cells or in solution as linkers, no cell spheroids were observed. After 96 h of incubation, the cultured DNA assembled spheroids were found to be mechanically stable enough to be handled easily for further analysis and confocal imaging. The findings suggest that the proposed DNA assembly method can be considered as an attractive strategy for assembling cells into stable spheroids.

Hydrophobically Modified Silica-Coated Gold Nanorods for Generating Nonlinear Photoacoustic Signals.

In this work, we report that gold nanorods coated with hydrophobically-modified mesoporous silica shells not only enhance photoacoustic (PA) signal over unmodified mesoporous silica coated gold nanorods, but that the relationship between PA amplitude and input laser fluence is strongly nonlinear. Mesoporous silica shells of ~14 nm thickness and with ~3 nm pores were grown on gold nanorods showing near infrared absorption. The silica was rendered hydrophobic with addition of dodecyltrichlorosilane, then re-suspended in aqueous media with a lipid monolayer. Analysis of the PA signal revealed not only an enhancement of PA signal compared to mesoporous silica coated gold nanorods at lower laser fluences, but also a nonlinear relationship between PA signal and laser fluence. We attribute each effect to the entrapment of solvent vapor in the mesopores: the vapor has both a larger expansion coefficient and thermal resistance than silica that enhances conversion to acoustic energy, and the hydrophobic porous surface is able to promote phase transition at the surface, leading to a nonlinear PA response even at fluences as low as 5 mJ cm-2. At 21 mJ cm-2, the highest laser fluence tested, the PA enhancement was >12-fold over mesoporous silica coated gold nanorods.

Investigating the use of conducting oligomers and redox molecules in CdS-MoFeP biohybrids.

In this work we report the effect of incorporating conducting oligophenylenes and a cobaltocene-based redox mediator on photodriven electron transfer between thioglycolic acid (TGA) capped CdS nanorods (NR) and the native nitrogenase MoFe protein (MoFeP) by following the reduction of H+ to H2. First, we demonstrate that the addition of benzidine-a conductive diphenylene- to TGA-CdS and MoFeP increased catalytic activity by up to 3-fold as compared to CdS-MoFeP alone. In addition, in comparing the use of oligophenylenes composed of one (p-phenylenediamine), two (benzidine) or three (4,4''-diamino-p-terphenyl)phenylene groups, the largest gain in H2 was observed with the addition of benzidine and the lowest with phenylenediamine. As a comparison to the conductive oligophenylenes, a cobaltocene-based redox mediator was also tested with the TGA-CdS NRs and MoFeP. However, adding either cobaltocene diacid or diamine caused negligible gains in H2 production and at higher concentrations, caused a significant decrease. Agarose gel electrophoresis revealed little to no detectable interaction between benzidine and TGA-CdS but strong binding between cobaltocene and TGA-CdS. These results suggest that the tight binding of the cobaltocene mediator to CdS may hinder electron transfer between CdS and MoFe and cause the mediator to undergo continuous reduction/oxidation events at the surface of CdS.

Nongenetic Bioconjugation Strategies for Modifying Cell Membranes and Membrane Proteins: A Review.

The cell membrane possesses an extensive library of proteins, carbohydrates, and lipids that control a significant portion of inter- and intracellular functions, including signaling, proliferation, migration, and adhesion, among others. Augmenting the cell surface composition would open possibilities for advances in therapy, tissue engineering, and probing fundamental cell processes. While genetic engineering has proven effective for many in vitro applications, these techniques result in irreversible changes to cells and are difficult to apply in vivo. Another approach is to instead attach exogenous functional groups to the cell membrane without changing the genetic nature of the cell. This review focuses on more recent approaches of nongenetic methods of cell surface modification through metabolic pathways, anchorage by hydrophobic interactions, and chemical conjugation. Benefits and drawbacks of each approach are considered, followed by a discussion of potential applications for nongenetic cell surface modification and an outlook on the future of the field.

Efficient cellular uptake of click nucleic acid modified proteins.

Efficient intracellular delivery of biomacromolecules such as proteins continues to remain a challenge despite its potential for medicine. In this work, we show that mScarlet, a non cytotoxic red fluorescent protein (RFP) conjugated to Click Nucleic Acid (CNA), a synthetic analog of DNA, undergo cell uptake significantly more than either native proteins or proteins conjugated with similar amounts of DNA in MDA-MB-468 cells. We further demonstrate that the process of cell uptake is metabolically driven and that scavenger receptors and caveolae mediated endocytosis play a significant role. Co-localization studies using anti-scavenger receptor antibodies suggest that scavenger receptors are implicated in the mechanism of uptake of CNA modified proteins.

Investigating Protein-Nanocrystal Interactions for Photodriven Activity.

We illustrate how intermolecular interactions facilitate ATP-free electron transfer between either native or engineered MoFe protein (MoFeP) from nitrogenase and a CdS nanorod (NR) by following the reduction of H+ to H2. First, by varying the charge on the surface of the NR, we show the role of electrostatic interactions on MoFeP binding to the particle surface and subsequent H+ reduction. Next, the role of strong, semicovalent thiol-CdS interactions was tested using free cysteines on the MoFeP. By blocking free cysteines, we show that the presence of free thiols on the protein has little to no influence on CdS binding and resultant photocatalytic activity. We next studied methods to covalently bind the protein to CdS by modifying the free cysteines with dibenzocyclooctyne (DBCO) and reacting the CdS NRs capped with a mixture of negatively charged thioglycolic acid and thiol-PEG3-azide ligands. As compared to that of the unmodified proteins, a 32.2 ± 1.5% and 61.7 ± 2.1% increase in H2 production was observed from MoFeP and C-MoFeP, respectively. At last, to test the effect of both charge and covalent tethering, positively charged cysteamine/azide CdS NRs were reacted with DBCO-modified C-MoFeP, which showed little improvement over native C-MoFeP alone under irradiation. These results show the importance of both electrostatic associations between the NR and protein and covalently tethering the protein to the semiconductor surface for enhanced electron transfer and photodriven activity.

Enzymes Photo-Cross-Linked to Live Cell Receptors Retain Activity and EGFR Inhibition after Both Internalization and Recycling.

In this work, we show that a prodrug enzyme covalently photoconjugated to live cell receptors survives endosomal proteolysis and retains its catalytic activity over multiple days. Here, a fusion protein was designed with both an antiepidermal growth factor receptor (EGFR) affibody and the prodrug enzyme cytosine deaminase, which can convert prodrug 5-fluorocytosine to the anticancer drug 5-fluorouracil. A benzophenone group was added at a site-specific mutation within the affibody, and the fusion protein was selectively photoconjugated to EGFR receptors expressed on membranes of MDA-MB-468 breast cancer cells. The fusion protein was next labeled with two dyes for tracking uptake: AlexaFluor 488 and pH-sensitive pHAb. Flow cytometry showed that fusion proteins photo-cross-linked to EGFR first underwent receptor-mediated endocytosis within 12 h, followed by recycling back to the cell membrane within 24 h. These findings were also confirmed by confocal microscopy. The unique cross-linking of the affibody-enzyme fusion proteins was utilized for two anticancer treatments. First, the covalent linking of the protein to the EGFR led to inhibition of ERK signaling over a two-day period, whereas conventional antibody therapy only led to 6 h of inhibition. Second, when the affibody-CodA fusion proteins were photo-cross-linked to EGFR overexpressed on MDA-MB-468 breast cancer cells, prodrug conversion was found even 48 h postincubation without any apparent decrease in cell killing, while without photo-cross-linking no cell killing was observed 8 h postincubation. These studies show that affinity-mediated covalent conjugation of the affibody-enzymes to cell receptors allows for prolonged expression on membranes and retained enzymatic activity without genetic engineering.

Surface-Templated Nanobubbles Protect Proteins from Surface-Mediated Denaturation.

In this Letter, we report that surface-bound nanobubbles reduce protein denaturation on methylated glass by irreversible protein shell formation. Single-molecule total internal reflection fluorescence (SM-TIRF) microscopy was combined with intramolecular Förster resonance energy transfer (FRET) to study the conformational dynamics of nitroreductase (NfsB) on nanobubble-laden methylated glass surfaces, using reflection brightfield microscopy to register nanobubble locations with NfsB adsorption. First, NfsB adsorbed irreversibly to nanobubbles with no apparent desorption after 5 h. Moreover, virtually all (96%) of the NfsB molecules that interacted with nanobubbles remained folded, whereas less than 50% of NfsB molecules remained folded in the absence of nanobubbles on unmodified silica or methylated glass surfaces. This trend was confirmed by ensemble-average fluorometer TIRF experiments. We hypothesize that nanobubbles reduce protein damage by passivating strongly denaturing topographical surface defects. Thus, nanobubble stabilization on surfaces may have important implications for antifouling surfaces and improving therapeutic protein storage.

Click Nucleic Acid Mediated Loading of Prodrug Activating Enzymes in PEG-PLGA Nanoparticles for Combination Chemotherapy.

The simultaneous delivery of multiple therapeutics to a single site has shown promise for cancer targeting and treatment. However, because of the inherent differences in charge and size between drugs and biomolecules, new approaches are required for colocalization of unlike components in one delivery vehicle. In this work, we demonstrate that triblock copolymers containing click nucleic acids (CNAs) can be used to simultaneously load a prodrug enzyme (cytosine deaminase, CodA) and a chemotherapy drug (doxorubicin, DOX) in a single polymer nanoparticle. CNAs are synthetic analogs of DNA comprised of a thiolene backbone and nucleotide bases that can hybridize to complementary strands of DNA. In this study, CodA was appended with complementary DNA sequences and fluorescent dyes to allow its encapsulation in PEG-CNA-PLGA nanoparticles. The DNA-modified CodA was found to retain its enzyme activity for converting prodrug 5-fluorocytosine (5-FC) to active 5-fluorouracil (5-FU) using a modified fluorescent assay. The DNA-conjugated CodA was then loaded into the PEG-CNA-PLGA nanoparticles and tested for cell cytotoxicity in the presence of the 5-FC prodrug. To study the effect of coloading DOX and CodA within a single nanoparticle, cell toxicity assays were run to compare dually loaded nanoparticles with nanoparticles loaded only with either DOX or CodA. We show that the highest level of cell death occurred when both DOX and CodA were simultaneously entrapped and delivered to cells in the presence of 5-FC.

Solar Photocatalytic Phenol Polymerization and Hydrogen Generation for Flocculation of Wastewater Impurities.

Achieving global sustainability will require balancing encroaching climate changes while maintaining existing quality of life. Using sunlight to purify wastewater while simultaneously generating usable fuels is an opportunity to approach both targets in a cost-efficient manner. In addition, converting biomass products to usable polymers is a sustainable approach for potentially replacing polystyrene or other petroleum derived polymers. Phenols from medical, manufacturing, and agricultural waste are commonly found in many water sources, and they are known to foul common reverse osmosis membranes. Here, we show oxidative polymerization of guaiacol, an aromatic compound derived from biomass, with concurrent hydrogen gas generation by using platinum-seeded cadmium sulfide nanorods (Pt@CdS) as photocatalysts. Rather than forming short oligomers as typically made by enzymes such as laccase and peroxidase, the resulting polymers show higher molecular weights that can more easily flocculate out of water. By comparing guaiacol conversion to molecular weight and dispersity, the guaiacol was found to polymerize via a chain-growth process. We also show that Pt@CdS can polymerize other phenols as well by testing the monomers phenol, 2,6-dihydroxybenzoic acid, gallic acid, and vanillin. Lastly, because the aqueous solubility of these aromatic polymers decreases dramatically with molecular weight, polymerization reactions were also tested in biphasic solutions to determine if chain growth could propagate in the oil phase. We show that the Pt@CdS nanoparticles can form stable Pickering emulsions in various biphasic combinations, and that both H2 formation and polymer molecular weight correlated with the partition coefficient of guaiacol into the oil phase as well as the solubility of the growing polymer chains. These combined studies demonstrate the possibility of using nanoscale photocatalysts to oxidatively polymerize phenolic substrates via a chain-growth mechanism, thereby providing a path for pretreating water by flocculating out contaminants with concurrent generation of hydrogen.

Nanoparticle-Mediated Acoustic Cavitation Enables High Intensity Focused Ultrasound Ablation Without Tissue Heating.

While thermal ablation of various solid tumors has been demonstrated using high intensity focused ultrasound (HIFU), the therapeutic outcomes of this technique are still unsatisfactory because of common recurrence of thermally ablated cancers and treatment side effects due to the high ultrasound intensity and acoustic pressure requirements. More precise ablation of tumors can be achieved by generating cavitating bubbles in the tissue using shorter pulses with higher acoustic pressures, which induce mechanical damage rather than thermal. However, it has remained as a challenge to safely deliver the acoustic pressures required for mechanical ablation of solid tumors. Here, we report a method to achieve mechanical ablation at lower acoustic pressures by utilizing phospholipid-stabilized hydrophobic mesoporous silica nanoparticles (PL-hMSN). The PL-hMSNs act as seeds for nucleation of cavitation events and thus significantly reduce the peak negative pressures and spatial-average temporal-average HIFU intensities needed to achieve mechanical ablation. Substantial mechanical damage was observed in the red blood cell or tumor spheroid containing tissue mimicking phantoms at PL-hMSN concentrations as low as 10 µg mL-1, after only 5 s of HIFU treatment with peak negative pressures ∼11 MPa and duty cycles ∼0.01%. Even the application of HIFU (peak negative pressure of 16.8 MPa and duty cycle of 0.017%) for 1 min in the presence of PL-hMSN (200 µg mL-1) did not cause any detectable temperature increase in tissue-mimicking phantoms. In addition, the mechanical effects of cavitation promoted by PL-hMSNs were observed up to 0.5 mm from the center of the cavitation events. This method may thus also improve delivery of therapeutics or nanoparticles to tumor environments with limited macromolecular transport.

New Generation of Clickable Nucleic Acids: Synthesis and Active Hybridization with DNA.

Due to the ability to generate oligomers of precise sequence, sequential and stepwise solid-phase synthesis has been the dominant method of producing DNA and other oligonucleotide analogues. The requirement for a solid support, however, and the physical restrictions of limited surface area thereon significantly diminish the efficiency and scalability of these syntheses, thus, negatively affecting the practical applications of synthetic polynucleotides and other similarly created molecules. By employing the robust photoinitiated thiol-ene click reaction, we developed a new generation of clickable nucleic acids (CNAs) with a polythioether backbone containing repeat units of six atoms, matching the spacing of the phosphodiester backbone of natural DNA. A simple, inexpensive, and scalable route was utilized to produce CNA monomers in gram-scale, which indicates the potential to dramatically lower the cost of these DNA mimics and thereby expand the scope of these materials. The efficiency of this approach was demonstrated by the completion of CNA polymerization in 30 seconds, as characterized by size-exclusive chromatography (SEC) and infrared (IR) spectroscopy. CNA/DNA hybridization was demonstrated by gel electrophoresis and used in CdS nanoparticle assembly.

Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors.

A significant challenge for solid tumor treatment is ensuring that a sufficient concentration of therapeutic agent is delivered to the tumor site at doses that can be tolerated by the patient. Biomolecular targeting can bias accumulation in tumors by taking advantage of specific interactions with receptors overexpressed on cancerous cells. However, while antibody-based immunoconjugates show high binding to specific cells, their low dissociation constants ( KD) and large Stokes radii hinder their ability to penetrate deep into tumor tissue, leading to incomplete cell killing and tumor recurrence. To address this, we demonstrate the design and production of a photo-cross-linkable affibody that can form a covalent bond to epidermal growth factor receptor (EGFR) under near UV irradiation. Twelve cysteine mutations were created of an EGFR affibody and conjugated with maleimide-benzophenone. Of these only one exhibited photoconjugation to EGFR, as demonstrated by SDS-PAGE and Western blot. Next this modified affibody was shown to not only bind EGFR expressing cells but also show enhanced retention in a 3D tumor spheroid model, with minimal loss up to 24 h as compared to either unmodified EGFR-binding affibodies or nonbinding, photo-cross-linkable affibodies. Finally, in order to show utility of photo-cross-linking at clinically relevant wavelengths, upconverting nanoparticles (UCNPs) were synthesized that could convert 980 nm light to UV and blue light. In the presence of UCNPs, both direct photoconjugation to EGFR and enhanced retention in tumor spheroids could be obtained using near-infrared illumination. Thus, the photoactive affibodies developed here may be utilized as a platform technology for engineering new therapy conjugates that can penetrate deep into tumor tissue and be retained long enough for effective tumor therapy.

TiO2-Capped Gold Nanorods for Plasmon-Enhanced Production of Reactive Oxygen Species and Photothermal Delivery of Chemotherapeutic Agents.

Near infrared (NIR)-absorbing noble metal nanostructures are being extensively studied as theranostic agents, in particular for photoacoustic imaging and photothermal therapy. Because of the electric field enhancement at the tips of anisotropic metal nanostructures, positioning photoactive species at these sites can lead to increased energy absorption. Herein, we show the site-specific placement of NIR-active photosensitizers at the ends of gold nanorods (AuNRs) by growing porous TiO2 caps. The surface plasmon resonance of the AuNRs was carefully tuned to overlap with the exciton absorption of indocyanine green (ICG), a NIR photosensitizer with low quantum yields and poor photostability. In conjugating high amounts of ICG to the TiO2 caps, increased amounts of singlet oxygen (1O2) were generated as compared to when ICG was attached to sidewalls of the AuNRs. Because the AuNRs also cause local increases in temperature upon NIR excitation, DNA strands were next attached to the AuNRs sidewalls and loaded with doxorubicin (DOX). We found that the synergistic effect of increased 1O2 and photothermal-induced drug delivery led to significant improvements in tumor cell killing. This work demonstrates that with careful design over hybrid nanostructure synthesis, higher levels of tumor therapy may be achieved.

Water-soluble clickable nucleic acid (CNA) polymer synthesis by functionalizing the pendant hydroxyl.

Synthetic biomacromolecules that mimic natural polymeric structures are of significant interest. For most applications of these materials, however, aqueous solubility is a necessity. Here, we present the synthesis of an intrinsically water soluble single stranded DNA analog formed by the synthesis of a Clickable Nucleic Acid (CNA). These molecules are formed with pendant hydroxyl groups present on the main polymer backbone, and subsequent modification of those hydroxyls with sulfonate moieties further enhances the hydrophilicity of these molecules.

DNA-Assembled Core-Satellite Upconverting-Metal-Organic Framework Nanoparticle Superstructures for Efficient Photodynamic Therapy.

DNA-mediated assembly of core-satellite structures composed of Zr(IV)-based porphyrinic metal-organic framework (MOF) and NaYF4 ,Yb,Er upconverting nanoparticles (UCNPs) for photodynamic therapy (PDT) is reported. MOF NPs generate singlet oxygen (1 O2 ) upon photoirradiation with visible light without the need for additional small molecule, diffusional photosensitizers such as porphyrins. Using DNA as a templating agent, well-defined MOF-UCNP clusters are produced where UCNPs are spatially organized around a centrally located MOF NP. Under NIR irradiation, visible light emitted from the UCNPs is absorbed by the core MOF NP to produce 1 O2 at significantly greater amounts than what can be produced from simply mixing UCNPs and MOF NPs. The MOF-UCNP core-satellite superstructures also induce strong cell cytotoxicity against cancer cells, which are further enhanced by attaching epidermal growth factor receptor targeting affibodies to the PDT clusters, highlighting their promise as theranostic photodynamic agents.