Biotargeted nanomedicines for cancer: six tenets before you begin.

Biotargeted nanomedicines have captured the attention of academic and industrial scientists who have been motivated by the theoretical possibilities of the 'magic bullet' that was first conceptualized by Paul Ehrlich at the beginning of the 20th century. The Biotargeting Working Group, consisting of more than 50 pharmaceutical scientists, engineers, biologists and clinicians, has been formed as part of the National Cancer Institute's Alliance for Nanotechnology in Cancer to harness collective wisdom in order to tackle conceptual and practical challenges in developing biotargeted nanomedicines for cancer. In modern science and medicine, it is impossible for any individual to be an expert in every aspect of biology, chemistry, materials science, pharmaceutics, toxicology, chemical engineering, imaging, physiology, oncology and regulatory affairs. Drawing on the expertise of leaders from each of these disciplines, this commentary highlights six tenets of biotargeted cancer nanomedicines in order to enable the translation of basic science into clinical practice.

Dendrimer-induced leukocyte procoagulant activity depends on particle size and surface charge.

AIMS: Thrombogenicity associated with the induction of leukocyte procoagulant activity (PCA) is a common complication in sepsis and cancer. Since nanoparticles are increasingly used for drug delivery, their interaction with coagulation systems is an important part of the safety assessment. The purpose of this study was to investigate the effects of nanoparticle physicochemical properties on leukocyte PCA, and to get insight into the mechanism of PCA induction. MATERIALS & METHODS: A total of 12 formulations of polyamidoamine (PAMAM) dendrimers, varying in size and surface charge, were studied in vitro using recalcification time assay. RESULTS: Irrespective of their size, anionic and neutral dendrimers did not induce leukocyte PCA in vitro. Cationic particles induced PCA in a size- and charge-dependent manner. The mechanism of PCA induction was similar to that of doxorubicin. Cationic dendrimers were also found to exacerbate endotoxin-induced PCA. CONCLUSION: PAMAM dendrimer-induced leukocyte PCA depends on particle size, charge and density of surface groups.

Nanoparticle size and surface charge determine effects of PAMAM dendrimers on human platelets in vitro.

Blood platelets are essential in maintaining hemostasis. Various materials can activate platelets and cause them to aggregate. Platelet aggregation in vitro is often used as a marker for materials' thrombogenic properties, and studying nanomaterial interaction with platelets is an important step toward understanding their hematocompatibility. Here we report evaluation of 12 formulations of PAMAM dendrimers varying in size and surface charge. Using a cell counter based method, light transmission aggregometry and scanning electron microscopy, we show that only large cationic dendrimers, but not anionic, neutral or small cationic dendrimers, induce aggregation of human platelets in plasma in vitro. The aggregation caused by large cationic dendrimers was proportional to the number of surface amines. The observed aggregation was not associated with membrane microparticle release, and was insensitive to a variety of chemical and biological inhibitors known to interfere with various pathways of platelet activation. Taken in context with previously reported studies, our data suggest that large cationic PAMAM dendrimers induce platelet aggregation through disruption of membrane integrity.

Translational considerations for cancer nanomedicine.

There are many important considerations during preclinical development of cancer nanomedicines, including: 1) unique aspects of animal study design; 2) the difficulties in evaluating biological potency, especially for complex formulations; 3) the importance of analytical methods that can determine platform stability in vivo, and differentiate bound and free active pharmaceutical ingredient (API) in biological matrices; and 4) the appropriateness of current dose scaling techniques for estimation of clinical first-in-man dose from preclinical data. Biologics share many commonalities with nanotechnology products with regard to complexity and biological attributes, and can, in some cases, provide context for dealing with these preclinical issues. In other instances, such as the case of in vivo stability analysis, new approaches are required. This paper will discuss the significance of these preclinical issues, and present examples of current methods and best practices for addressing them. Where possible, these recommendations are justified using the existing regulatory guidance literature.

Nanomaterial standards for efficacy and toxicity assessment.

Decreased toxicity via selective delivery of cancer therapeutics to tumors has become a hallmark achievement of nanotechnology. In order to be optimally efficacious, a systemically administered nanomedicine must reach cancer cells in sufficient quantities to elicit a response and assume its active form within the tumor microenvironment (e.g., be taken up by cancer cells and release a toxic component once within the cytosol or nuclei). Most nanomedicines achieve selective tumor accumulation via the enhanced permeability and retention (EPR) effect or a combination of the EPR effect and active targeting to cellular receptors. Here, we review how the fundamental physicochemical properties of a nanomedicine (its size, charge, hydrophobicity, etc.) can dramatically affect its distribution to cancerous tissue, transport across vascular walls, and retention in tumors. We also discuss how nanoparticle characteristics such as stability in the blood and tumor, cleavability of covalently bound components, cancer cell uptake, and cytotoxicity contribute to efficacy once the nanoparticle has reached the tumor's interstitial space. We elaborate on how tumor vascularization and receptor expression vary depending on cancer type, stage of disease, site of implantation, and host species, and review studies which have demonstrated that these variations affect tumor response to nanomedicines. Finally, we show how knowledge of these properties (both of the nanoparticle and the cancer/tumor under study) can be used to design meaningful in vivo tests to evaluate nanoparticle efficacy.

Nanoparticle interaction with plasma proteins as it relates to particle biodistribution, biocompatibility and therapeutic efficacy.

Proteins bind the surfaces of nanoparticles, and biological materials in general, immediately upon introduction of the materials into a physiological environment. The further biological response of the body is influenced by the nanoparticle-protein complex. The nanoparticle's composition and surface chemistry dictate the extent and specificity of protein binding. Protein binding is one of the key elements that affects biodistribution of the nanoparticles throughout the body. Here we review recent research on nanoparticle physicochemical properties important for protein binding, techniques for isolation and identification of nanoparticle-bound proteins, and how these proteins can influence particle biodistribution and biocompatibility. Understanding the nanoparticle-protein complex is necessary for control and manipulation of protein binding, and allows for improved engineering of nanoparticles with favorable bioavailability and biodistribution.

Interaction of colloidal gold nanoparticles with human blood: effects on particle size and analysis of plasma protein binding profiles.

Nanoparticle size and plasma binding profile contribute to a particle's longevity in the bloodstream, which can have important consequences for therapeutic efficacy. In this study an approximate doubling in nanoparticle hydrodynamic size was observed upon in vitro incubation of 30- and 50-nm colloidal gold in human plasma. Plasma proteins that bind the surface of citrate-stabilized gold colloids have been identified. Effects of protein binding on the nanoparticle hydrodynamic size, elements of coagulation, and the complement system have been investigated. The difference in size measurements obtained from dynamic light scattering, electron microscopy, and scanning probe microscopy are also discussed.

Method for analysis of nanoparticle hemolytic properties in vitro.

Hemolysis (destruction of red blood cells) in vivo can lead to anemia, jaundice, and other pathological conditions; therefore the hemolytic potential of all intravenously administered pharmaceuticals must be evaluated. Nanotechnology-derived devices and drug carriers are emerging as alternatives to conventional small-molecule drugs, and in vitro evaluation of their biocompatibility with blood components is a necessary part of early preclinical development. The small size and unique physicochemical properties of nanoparticles may cause their interactions with erythrocytes to differ from those observed for conventional pharmaceuticals and may also cause interference with standardized in vitro tests. Separating true hemolytic responses from the false-positive or false-negative results caused by particle interference is important for correct interpretation of these tests. Here we describe validation of an in vitro assay for the analysis of nanoparticle hemolytic properties and discuss observed nanointerferences with the assay. We propose alternative methods to avoid misleading results from nanoparticles and discuss the potential relevance of nanoparticle in vitro hemolytic properties to in vivo systems.

Preclinical studies to understand nanoparticle interaction with the immune system and its potential effects on nanoparticle biodistribution.

Nanoparticles have unique physicochemical properties which make them promising platforms for drug delivery. However, immune cells in the bloodstream (such as monocytes, platelets, leukocytes, and dendritic cells) and in tissues (such as resident phagocytes) have a propensity to engulf and eliminate certain nanoparticles. A nanoparticle's interaction with plasma proteins (opsonins) and blood components (via hemolysis, thrombogenicity and complement activation) may influence uptake and clearance and hence potentially affect distribution and delivery to the intended target sites. Nanoparticle uptake by the immune cells is influenced by many factors. Different nanoparticles have been shown to act on different pathways, while various characteristics/properties also affect which pathway is employed for particle internalization. Nanoparticle protein binding occurs almost instantaneously once the particle enters biological medium, and the physical properties of such a particle-protein complex are often different than those of the formulated particle. These new properties can contribute to different biological responses and change nanoparticle biodistribution. Therefore, in the situation when specific delivery to immune cells is not desired, the ideal nanoparticle platform is the one whose integrity is not disturbed in the complex biological environment, which provides extended circulation in the blood to maximize delivery to the target site, is not toxic to blood cellular components, and is "invisible" to the immune cells which can remove it from circulation. This review discusses the most recent data on nanoparticle interactions with blood components and how particle size and surface charge define their hematocompatibility. This includes properties which determine particle interaction with plasma proteins and uptake by macrophages. We will also provide an overview of in vitro methods useful in identifying interactions with components of the immune system and the potential effects of such interaction on particle distribution to tissues.

Characterization of nanoparticles for therapeutics.

Nanotechnology offers many advantages to traditional drug design, delivery and medical diagnostics; however, nanomedicines present considerable challenges for preclinical development. Nanoparticle constructs intended for medical applications consist of a wide variety of materials, and their small size, unique physicochemical properties and biological activity often require modification of standard characterization techniques. A rational characterization strategy for nanomedicines includes physicochemical characterization, sterility and pyrogenicity assessment, biodistribution (absorption, distribution, metabolism and excretion [ADME]) and toxicity characterization, which includes both in vitro tests and in vivo animal studies. Here, we highlight progress for a few methods that are uniquely useful for nanoparticles or are indicative of their toxicity or efficacy.

Measurement of 15N relaxation in deuterated amide groups in proteins using direct nitrogen detection.

15N chemical shielding tensors contain useful structural information, and their knowledge is essential for accurate analysis of protein backbone dynamics. The anisotropic component (CSA) of 15N chemical shielding can be obtained from 15N relaxation measurements in solution. However, the predominant contribution to nitrogen relaxation from 15N-(1)H dipolar coupling in amide groups limits the sensitivity of these measurements to the actual CSA values. Here we present nitrogen-detected NMR experiments for measuring 15N relaxation in deuterated amide groups in proteins, where the dipolar contribution to 15N relaxation is significantly reduced by the deuteration. Under these conditions nitrogen spin relaxation becomes a sensitive probe for variations in 15N chemical shielding tensors. Using the nitrogen direct-detection experiments we measured the rates of longitudinal and transverse 15N relaxation for backbone amides in protein G in D(2)O at 11.7 T. The measured relaxation rates are validated by comparing the overall rotational diffusion tensor obtained from these data with that from the conventional 15N relaxation measurements in H(2)O. This analysis revealed a 17-24 degree angle between the NH-bond and the unique axis of the 15N chemical shielding tensor.

Variability of the 15N chemical shielding tensors in the B3 domain of protein G from 15N relaxation measurements at several fields. Implications for backbone order parameters.

We applied a combination of 15N relaxation and CSA/dipolar cross-correlation measurements at five magnetic fields (9.4, 11.7, 14.1, 16.4, and 18.8 T) to determine the 15N chemical shielding tensors for backbone amides in protein G in solution. The data were analyzed using various model-independent approaches and those based on Lipari-Szabo approximation, all of them yielding similar results. The results indicate a range of site-specific values of the anisotropy (CSA) and orientation of the 15N chemical shielding tensor, similar to those in ubiquitin (Fushman, et al. J. Am. Chem. Soc. 1998, 120, 10947; J. Am. Chem. Soc. 1999, 121, 8577). Assuming a Gaussian distribution of the 15N CSA values, the mean anisotropy is -173.9 to -177.2 ppm (for 1.02 A NH bond length) and the site-to-site CSA variability is +/-17.6 to +/-21.4 ppm, depending on the method used. This CSA variability is significantly larger than derived previously for ribonuclease H (Kroenke, et al. J. Am. Chem. Soc. 1999, 121, 10119) or recently, using "meta-analysis" for ubiquitin (Damberg, et al. J. Am. Chem. Soc. 2005, 127, 1995). Standard interpretation of 15N relaxation studies of backbone dynamics in proteins involves an a priori assumption of a uniform 15N CSA. We show that this assumption leads to a significant discrepancy between the order parameters obtained at different fields. Using the site-specific CSAs obtained from our study removes this discrepancy and allows simultaneous fit of relaxation data at all five fields to Lipari-Szabo spectral densities. These findings emphasize the necessity of taking into account the variability of 15N CSA for accurate analysis of protein dynamics from 15N relaxation measurements.

Spin-state selection for increased confidence in cross-correlation rates measurements.

A new approach is described for measuring chemical shift anisotropy (CSA)/dipolar cross-correlated relaxation (CCR) rates based on the selection of the individual 15N doublet components prior to the relaxation period. The method uses the spin-state-selective element (S3E) of Sørensen and co-authors [Meissner et al. (1997) J. Mag. Reson., 128, 92-97]. The main advantage of the new method compared to other J-resolved experiments is that it does not create problems of additional signal overlap encountered in coupled spectra. At the same time, this approach allows a simpler control of magnetization pathways than the indirect methods. The method is demonstrated for the B3 domain of protein G.

Characterization of the overall and local dynamics of a protein with intermediate rotational anisotropy: Differentiating between conformational exchange and anisotropic diffusion in the B3 domain of protein G.

Because the overall tumbling provides a major contribution to protein spectral densities measured in solution, the choice of a proper model for this motion is critical for accurate analysis of protein dynamics. Here we study the overall and backbone dynamics of the B3 domain of protein G using (15)N relaxation measurements and show that the picture of local motions is markedly dependent on the model of overall tumbling. The main difference is in the interpretation of the elevated R(2) values in the alpha-helix: the isotropic model results in conformational exchange throughout the entire helix, whereas no exchange is predicted by anisotropic models that place the longitudinal axis of diffusion tensor almost parallel to the helix axis. Due to small size (fast tumbling) of the protein, the T(1) values have low sensitivity to NH bond orientation. The diffusion tensor derived from orientation dependence of R(2)/R(1) is anisotropic (D(par)/D(perp)=1.4), with a small rhombic component. In order to distinguish the correct picture of motion, we apply model-independent methods that are sensitive to conformational exchange and do not require knowledge of protein structure or assumptions about its dynamics. A comparison of the CSA/dipolar cross-correlation rate constants with (15)N relaxation rates and the estimation of R(ex) terms from relaxation data at 9.4 and 14.1 T indicate no conformational exchange in the helix, in support of the anisotropic models. The experimentally derived diffusion tensor is in excellent agreement with theoretical predictions from hydrodynamic calculations; a detailed comparison with various hydrodynamic models revealed optimal parameters for hydrodynamic calculations.

Direct measurement of the 15N CSA/dipolar relaxation interference from coupled HSQC spectra.

Here we propose a method for the measurement of the (15)N CSA/dipolar relaxation interference based on direct comparison of the (15)N doublet components observed in a (1)H-coupled (1)H-(15)N HSQC-type spectrum. This allows the determination of the cross-correlation rates with no need for correction factors associated with other methods. The signal overlap problem of coupled HSQC spectra is addressed here by using the IPAP scheme (Ottiger et al., 1998). The approach is applied to the B3 domain of protein G to show that the method provides accurate measurements of the (15)N CSA/dipolar cross-correlation rates.