Incorporation of doxorubicin in different polymer nanoparticles and their anti-cancer activity

S Pieper, Hannah Onafuye, Dennis Mulac, Jindrich Cinatl, Mark N Wass, Martin Michaelis, Klaus Langer

Preprint posted on August 29, 2018

A whole new world: Characterising the release profile and biological efficacy of three doxorubicin-loaded nanoparticle formulations

Selected by Zhang-He Goh

Background of preprint: Not only dreaming

Doxorubicin is a chemotherapeutic drug commonly used to treat many cancers. Unfortunately, it is notable for its multiple toxicities—the most serious is cardiomyopathy, which is lethal enough to limit the maximum dose that patients can tolerate. In fact, the damage that doxorubicin and its fellow anthracycline analogues cause to patients’ hearts force healthcare professionals to restrict patients to maximum lifetime doses. The tragic implication of this is that anticancer regimens containing these anthracyclines may not be used again should the cancer recur, even if these drugs initially worked in patients.

Like all therapeutics, there are two classical ways that researchers can use to mitigate the problem posed by anthracycline toxicity. First, the effectiveness of anthracyclines in the general patient population can be increased by enhancing their potency. Second, the toxicity of these drugs can be reduced by increasing their selectivity.

By characterising the impact of incorporating doxorubicin in different polymer nanoparticles, Pieper et al look to achieve these two goals through the lens of drug delivery. This is a strategy employed by formulators that involves the use of specialised delivery methods to achieve these ends, instead of changing the structure of the drug in question. Specifically, Pieper et al aimed to use nanoparticle drug delivery systems to increase the specificity of doxorubicin delivery by exploiting the enhanced permeability and retention (EPR) effect, while also encouraging drug transport into cancer cells.

Key findings of preprint: A hundred thousand things to see

The experiments conducted by Pieper et al can be classified into two broad categories: (A) Preparation and characterisation of the doxorubicin-loaded polymer nanoparticles, and (B) the in vitro efficacy of these doxorubicin-loaded nanoparticles when compared with doxorubicin solution.

(A) Preparation and characterisation of the doxorubicin-loaded polymer nanoparticles

Pieper et al synthesised three preparations of polymer nanoparticles—polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA) nanoparticles, and PLGA-PEG nanoparticles—and characterised their particle size and size distribution.

Particle size. After testing the two methods of emulsion diffusion and solvent displacement in nanoparticle preparation, Pieper et al found that the PLGA nanoparticles yielded were of relatively similar mean sizes, while the PLGA-PEG nanoparticles yielded had markedly different mean sizes. Notably, the PLGA-PEG nanoparticles yielded by the solvent displacement technique were significantly smaller than the PLGA-PEG, PLGA, and PLA nanoparticles yielded by other techniques. Pieper et al also found that the size distribution of the particles synthesised were similar, with all of them exhibiting a monodisperse distribution.

Loading efficiency and drug release. Pieper et al sought to avoid burst release and enhance the loading efficiency of their nanoparticles. To these two ends, they successfully increased the loading efficiency of PLGA nanoparticles through an optimisation process. While all their nanoparticles exhibited burst release kinetics, they found that the PLGA-PEG nanoparticles were not as prone to burst release as was hypothesised based on evidence from existing literature [1].

(B) In vitro efficacy of these doxorubicin-loaded nanoparticles

Pieper et al found that among the preparations of nanoparticles tested, the PLGA nanoparticles prepared by solvent displacement at pH 7 and PLGA-PEG nanoparticles prepared by solvent displacement were the most efficacious, with activities similar to free doxorubicin in solution. Their efficacies were ascribed to two different mechanisms. The PLGA-PEG nanoparticles prepared by solvent displacement had the smallest size, and therefore exhibited the highest amount of uptake into cells. In contrast, the PLGA nanoparticles prepared by solvent displacement at pH 7 had the highest drug load because doxorubicin had the highest lipophilicity at this pH. This increased the amount of drug transported into cells per nanoparticle.

What I like about this preprint: Shining, shimmering, splendid

This preprint caught my attention for three main reasons. First and foremost, it addresses an important research gap. Toxicity ranks among the top reasons for failure of drug candidates in clinical trials. Even for more well-established drugs, such as the anticancer chemotherapeutics investigated in this preprint, there will always be a need to decrease toxicities for the lack of better alternatives. From this perspective, this preprint makes a key contribution to solving the problem of anthracycline toxicity.

Second, the findings of this preprint generate a slew of questions that can lead to future work in two fields. For one, the field of drug formulation benefits directly from the synthesis, optimisation, and characterisation of three different formulations of nanoparticles by Pieper et al. Moreover, Pieper et al also demonstrated that while other nanoparticle formulations bypassed efflux-mediated drug resistance, both the doxorubicin-loaded PLGA-PEG nanoparticles prepared by solvent displacement as well as the doxorubicin-loaded PLGA nanoparticles prepared by solvent displacement at pH 7 were unable to overcome transporter-mediated drug resistance mediated by the ABCB1 efflux transporter despite their enhanced efficacy.

Everything considered, these findings serve as a timely reminder that, as the famous physicist Feynman remarked, there is plenty of room at the bottom. Because each nanoparticle formulation has its own unique characteristics, the pharmacokinetics of nanoparticles are not easily profiled, and no single set of principles can be generalised across nanoparticle formulations. Even the EPR effect is controversial as at the time of posting this preLight [2], suggesting that much more research is necessary in understanding the roles and mechanisms that nanoparticles play in drug delivery before researchers better optimise and synthesise clinically useful nanoparticles.

The third reason for choosing this preprint is to highlight two of its most promising findings related to efficacy: The PLGA nanoparticles prepared by solvent displacement at pH 7 and PLGA-PEG nanoparticles prepared by solvent displacement. By identifying ways to 1) decrease the size of synthesised nanoparticles, and 2) to increase the drug load of each nanoparticle, more potent and selective drug delivery systems may be invented. At the same time, the entire optimisation process of preparing nanoparticles also provides valuable information on what works—and, equally importantly, what does not.

Future directions: An endless diamond sky

To paraphrase Aladdin, this preprint by Pieper et al is exciting because it opens new horizons for a broad spectrum of researchers to pursue. Based on the preliminary results offered by this preprint, formulators can subject formulations to a more rigorous and systematic process of optimisation over a larger range of conditions. Pharmacokineticists can profile the behaviour of these nanoparticle formulations in vitro and in vivo. Once the effectiveness and safety of these drug delivery systems are demonstrated in animal models, clinician-scientists can run in-human trials on patients who stand to gain from these drug delivery systems.

Professions other than scientists also stand to gain from future work on this preprint. Further research on the safety and efficacy profiles of these nano-sized delivery systems will paint a clearer picture of the benefits and risks to these formulations, guiding drug regulatory agencies around the world to make better decisions on whether to approve a given formulation. Should this work be successfully moved from the bench to the bedside, then clinicians and pharmacists, too, will have a greater arsenal of drugs at their disposal to treat patients better.

The field of nanotechnology is a dazzling place filled with wonders for engineers, nanotechnologists, and healthcare researchers and professionals alike. And, when these inventions are translated into novel drug discovery methods, patients will benefit from this whole new world too.

Questions for authors

  1. Given the controversy of the EPR effect, to what extent does this account for the increased uptake of smaller-sized PLGA nanoparticles by the neuroblastoma cell line compared to the larger-sized nanoparticle formulations?
  2. This preprint largely focuses on evaluating the efficacy of these doxorubicin-loaded nanoparticles on neuroblastoma cell lines. Have these formulations been tested on other endogenous cell lines, and if so, how did they perform in terms of toxicity? For example, doxorubicin is known to damage cardiomyocytes through mechanisms such as oxidative stress.
  3. Have these formulations been tested in vivo? If not, how did the observations in this preprint compare to similar formulations tested in vivo in the literature? Were differences in absorption, distribution, metabolism, and excretion observed compared to free doxorubicin solution, and were there any transporter-specific interactions, such as with ABCB1 that was highlighted in this preprint?


[1] Ruan G, Feng S-S, Preparation and characterization of poly(lactic acid)–poly(ethylene glycol)–poly(lactic acid) (PLA–PEG–PLA) microspheres for controlled release of paclitaxel, Biomaterials 24(27) (2003) 5037-5044.

[2] Youn YS, Bae YH, Perspectives on the past, present, and future of cancer nanomedicine, Advanced Drug Delivery Reviews 130 (2018) 3-11.

Tags: drug delivery, nanoparticles

Posted on: 19th September 2018

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