Yi-An Lin

Supramolecular nanostructures formed by anticancer drug assembly.

We report here a supramolecular strategy to directly assemble the small molecular hydrophobic anticancer drug camptothecin (CPT) into discrete, stable, well-defined nanostructures with a high and quantitative drug loading. Depending on the number of CPTs in the molecular design, the resulting nanostructures can be either nanofibers or nanotubes, and have a fixed CPT loading content ranging from 23% to 38%. We found that formation of nanostructures provides protection for both the CPT drug and the biodegradable linker from the external environment and thus offers a mechanism for controlled release of CPT. Under tumor-relevant conditions, these drug nanostructures can release the bioactive form of CPT and show in vitro efficacy against a number of cancer cell lines. This strategy can be extended to construct nanostructures of other types of anticancer drugs and thus presents new opportunities for the development of self-delivering drugs for cancer therapeutics.

Kinetic quantification of protein polymer nanoparticles using non-invasive imaging

Protein polymers are repetitive amino acid sequences that can assemble monodisperse nanoparticles with potential applications as cancer nanomedicines. Of the currently available molecular imaging methods, positron emission tomography (PET) is the most sensitive and quantitative; therefore, this work explores microPET imaging to track protein polymer nanoparticles over several days. To achieve reliable imaging, the polypeptides were modified by site-specific conjugation using a heterobifunctional sarcophagine chelator, AmBaSar, which was subsequently complexed with (64)Cu. AmBaSar/(64)Cu was selected because it can label particles in vivo over periods of days, which is consistent with the timescales required to follow long-circulating nanotherapeutics. Using an orthotopic model of breast cancer, we observed four elastin-like polypeptides (ELPs)-based protein polymers of varying molecular weight, amino acid sequence, and nanostructure. To analyze this data, we developed a six-compartment image-driven pharmacokinetic model capable of describing their distribution within individual subjects. Surprisingly, the assembly of an ELP block copolymer (78 kD) into nanoparticles (R(h) = 37.5 nm) minimally influences pharmacokinetics or tumor accumulation compared to a free ELP of similar length (74 kD). Instead, ELP molecular weight is the most important factor controlling the fate of these polymers, whereby long ELPs (74 kD) have a heart activity half-life of 8.7 hours and short ELPs (37 kD) have a half-life of 2.1 hours. These results suggest that ELP-based protein polymers may be a viable platform for the development of multifunctional therapeutic nanoparticles that can be imaged using clinical PET scanners.

Self-Assembled Tat Nanofibers as Effective Drug Carrier and Transporter

Cell penetrating peptides (CPPs) have been extensively explored as molecular vectors through covalent linkage to anticancer drugs to improve the drugs water solubility and to help overcome multidrug resistance. We report here the use of the Tat CPP as a molecular building unit to construct well-defined supramolecular nanofibers that can be utilized as a nanoscale vector to encapsulate the hydrophobic drug paclitaxel (PTX) (loading efficiency: 89.7 ± 5.0%) with a high loading capacity (6.8 ± 0.4%). Notably, our TEM imaging results reveal that nanofibers containing a higher PTX content tend to be more flexible than those with a lower PTX content. Fluorescence and confocal microscopy imaging show that the Tat nanofibers can effectively transport encapsulated molecules into the cells through an adsorptive-mediated endocytosis pathway. Cytotoxicity experiments and flow cytometry measurements demonstrate that PTX loaded in the nanofibers exerts its cytotoxicity against cancer cells by arresting the cells at the G2/M phase, the same working mechanism as free PTX.

Supramolecular filaments containing a fixed 41% paclitaxel loading

We report here the self-assembly of a rationally designed paclitaxel drug amphiphile into well-defined supramolecular filaments that possess a fixed 41% paclitaxel loading. These filaments can exert effective cytotoxicity against a number of cell lines comparable to that of free paclitaxel.

Elastin-based protein polymer nanoparticles carrying drug at both corona and core suppress tumor growth in vivo.

Numerous nanocarriers of small molecules depend on either non-specific physical encapsulation or direct covalent linkage. In contrast, this manuscript explores an alternative encapsulation strategy based on high-specificity avidity between a small molecule drug and its cognate protein target fused to the corona of protein polymer nanoparticles. With the new strategy, the drug associates tightly to the carrier and releases slowly, which may decrease toxicity and promote tumor accumulation via the enhanced permeability and retention effect. To test this hypothesis, the drug Rapamycin (Rapa) was selected for its potent anti-proliferative properties, which give it immunosuppressant and anti-tumor activity. Despite its potency, Rapa has low solubility, low oral bioavailability, and rapid systemic clearance, which make it an excellent candidate for nanoparticulate drug delivery. To explore this approach, genetically engineered diblock copolymers were constructed from elastin-like polypeptides (ELPs) that assemble small (<100nm) nanoparticles. ELPs are protein polymers of the sequence (Val-Pro-Gly-Xaa-Gly)n, where the identity of Xaa and n determine their assembly properties. Initially, a screening assay for model drug encapsulation in ELP nanoparticles was developed, which showed that Rose Bengal and Rapa have high non-specific encapsulation in the core of ELP nanoparticles with a sequence where Xaa=Ile or Phe. While excellent at entrapping these drugs, their release was relatively fast (2.2h half-life) compared to their intended mean residence time in the human body. Having determined that Rapa can be non-specifically entrapped in the core of ELP nanoparticles, FK506 binding protein 12 (FKBP), which is the cognate protein target of Rapa, was genetically fused to the surface of these nanoparticles (FSI) to enhance their avidity towards Rapa. The fusion of FKBP to these nanoparticles slowed the terminal half-life of drug release to 57.8h. To determine if this class of drug carriers has potential applications in vivo, FSI/Rapa was administered to mice carrying a human breast cancer model (MDA-MB-468). Compared to free drug, FSI encapsulation significantly decreased gross toxicity and enhanced the anti-cancer activity. In conclusion, protein polymer nanoparticles decorated with the cognate receptor of a high potency, low solubility drug (Rapa) efficiently improved drug loading capacity and its release. This approach has applications to the delivery of Rapa and its analogs; furthermore, this strategy has broader applications in the encapsulation, targeting, and release of other potent small molecules.

Rational design of MMP degradable peptide-based supramolecular filaments.

One-dimensional nanostructures formed by self-assembly of small molecule peptides have been extensively explored for use as biomaterials in various biomedical contexts. However, unlike individual peptides that can be designed to be specifically degradable by enzymes/proteases of interest, their self-assembled nanostructures, particularly those rich in β-sheets, are generally resistant to enzymatic degradation because the specific cleavage sites are often embedded inside the nanostructures. We report here on the rational design of β-sheet rich supramolecular filaments that can specifically dissociate into less stable micellar assemblies and monomers upon treatment with matrix metalloproteases-2 (MMP-2). Through linkage of an oligoproline segment to an amyloid-derived peptide sequence, we first synthesized an amphiphilic peptide that can undergo a rapid morphological transition in response to pH variations. We then used MMP-2 specific peptide substrates as multivalent cross-linkers to covalently fix the amyloid-like filaments in the self-assembled state at pH 4.5. Our results show that the cross-linked filaments are stable at pH 7.5 but gradually break down into much shorter filaments upon cleavage of the peptidic cross-linkers by MMP-2. We believe that the reported work presents a new design platform for the creation of amyloid-like supramolecular filaments responsive to enzymatic degradation.

Synthesis and Self-Assembly of a Mikto-Arm Star Dual Drug Amphiphile Containing both Paclitaxel and Camptothecin.

Self-assembly of anticancer therapeutics into discrete nanostructures provides an innovative way to develop a self-delivering nanomedicine with a high, quantitative drug loading. We report here the synthesis and assembly of a mikto-arm star dual drug amphiphile (DA) containing both a bulky paclitaxel (PTX) and a planar camptothecin (CPT). The two anti-cancer drugs of interest were stochastically conjugated to a β-sheet forming peptide (Sup35) and under physiologically-relevant conditions the dual DA could spontaneously associate into supramolecular filaments with a fixed 41% total drug loading (29% PTX and 12% CPT). Transmission electron microscopy imaging and circular dichroism spectroscopy studies reveal that the bulkiness of the PTX, as well as the π-π interaction preference between the CPT units, has a significant impact on the assembly kinetics, molecular level packing, and nanostructure morphology and stability. We found that the DA containing two PTX units assembled into non-filamentous micelle-like structures, in contrast to the filamentous structures formed by the hetero dual DA and the DA containing two CPTs. The hetero dual DA was found to effectively release the two anticancer agents, exhibiting superior cytotoxicity against PTX-resistant cervical cancer cells. The presented work offers a potential method to generate well-defined entwined filamentous nanostructures and provides the basis for a future combination therapy platform.

Controlled release of free doxorubicin from peptide–drug conjugates by drug loading

Covalent modification of a drug with a peptide moiety has been extensively used as an effective strategy to improve the drugs therapeutic outcome. One important consideration in the design of such a prodrug is the release of the free drug from the covalently bound form in a desired fashion. In most cases, the free drug release rate is controlled by the use of various chemical linkers that bridge the drug to the auxiliary segment. We report here that the degree of drug conjugation per peptide could also regulate the drug release in addition to its apparent effect on drug loading of the resulting conjugates. In this work, we synthesized three peptide-drug conjugates (NTD, d-NTD and q-NTD) in which the cell penetrating peptide Tat is covalently connected to one, two, or four doxorubicin, respectively, through a cathepsin B degradable tetrapeptide linker (-Gly-Phe-Leu-Gly-). We found that the number of doxorubicin within the conjugate impacts the release of doxorubicin in a significant way, with q-NTD showing the slowest release rate while NTD showing the fastest release rate. Our cellular uptake experiments reveal that q-NTD accumulated most effectively within cancer cells while NTD shows the lowest intracellular accumulation concentration. Interestingly, our cell viability assessment using a SRB assay reveals that d-NTD is the most potent conjugate against HepG2 human liver cancer cells. These results suggest that intracellular accumulation efficiency and the free drug release rate are two important factors that determine the in vitro efficacy of drug conjugates. To further validate this conclusion, we conjugated a short hydrocarbon onto the NTD to improve its cellular uptake, and found that the resulting conjugate, C16NTD, exhibited comparable intracellular accumulation as the q-NTD conjugate but superior anticancer activity due to its more effective release of free doxorubicin.

Self-assembly of natural and synthetic drug amphiphiles into discrete supramolecular nanostructures

Molecular assembly provides an effective approach to construct discrete supramolecular nanostructures of various sizes and shapes in a simple manner. One important technological application of the resulting nanostructures is their potential use as anticancer drug carriers to facilitate targeted delivery to tumour sites and consequently to improve clinical outcomes. In this carrier-assisted delivery strategy, anticancer drugs have been almost exclusively considered as the cargo to be carried and delivered, and their potential as molecular building blocks has been largely ignored. In this discussion, we report the use of anticancer drugs as molecular building units to create discrete supramolecular nanostructures that contain a high and quantitative drug loading and also have the potential for self-delivery. We first show the direct assembly of two amphiphilic drug molecules (methotrexate and folic acid) into discrete nanostructures. Our results reveal that folic acid exhibits rich self-assembly behaviour via Hoogsteen hydrogen bonding under various solvent conditions, whereas methotrexate is unable to assemble into any well-defined nanostructures under the same conditions, despite its similar chemical structure. Considering the low water solubility of most anticancer drugs, hydrophilic segments must be conjugated to the drug in order to bestow the necessary amphiphilicity. We have demonstrated this for camptothecin through the attachment of beta-sheet-forming peptides with overall hydrophilicity. We found that the intermolecular interactions among camptothecin segments and those among beta-sheet peptides act together to define the formation of stable one-dimensional nanostructures in dilute solutions, giving rise to nanotubes or nanofibers depending upon the processing conditions used. These results lead us to believe that self-assembly of drugs into discrete nanostructures not only offers an innovative way to craft self-delivering anticancer drugs, but also extends the paradigm of using molecular assembly as a toolbox to achieve functional nanostructures, to a new area which is specifically focused on the direct assembly of functional molecules (e.g. drugs, or imaging agents) into nanostructures of their own.

A quantitative recipe for engineering protein polymer nanoparticles

Protein polymers can assemble switchable nanostructures with emerging applications as biomaterials and nanomedicines. For example, above a critical micelle temperature (CMT) some elastin-like polypeptide (ELP) diblock copolymers assemble spherical nanoparticles, which may modulate cellular internalization and in vivo biodistribution. To achieve engineering-level control over their properties, this report explores a comprehensive library of ELP monoblock and diblock polymers. For the first time, we report that a surprisingly high core molecular weight is required for stable nanoparticle formation; furthermore, nanoparticle size depends on polymer molecular weight. A mathematical model was developed to characterize four ELP monoblock libraries and to predict the phase behavior of corresponding diblock copolymers. The CMT was almost entirely dependent on the hydrophobic core ELP, while the bulk phase transition temperature (Tt,bulk ) depends predominantly on the hydrophilic block. Nanoparticle assembly was accompanied by a conversion in secondary structure of the hydrophobic block from random coil and beta-sheets to type-2 β turns. For the first time, this report enables the rational design of ELP protein polymer nanoparticles with physico-chemico properties that will be suitable for biological applications.